Escherichia coli DNA replication: the old model organism still holds many surprises

Abstract Research on Escherichia coli DNA replication paved the groundwork for many breakthrough discoveries with important implications for our understanding of human molecular biology, due to the high level of conservation of key molecular processes involved. To this day, it attracts a lot of attention, partially by virtue of being an important model organism, but also because the understanding of factors influencing replication fidelity might be important for studies on the emergence of antibiotic resistance. Importantly, the wide access to high-resolution single-molecule and live-cell imaging, whole genome sequencing, and cryo-electron microscopy techniques, which were greatly popularized in the last decade, allows us to revisit certain assumptions about the replisomes and offers very detailed insight into how they work. For many parts of the replisome, step-by-step mechanisms have been reconstituted, and some new players identified. This review summarizes the latest developments in the area, focusing on (a) the structure of the replisome and mechanisms of action of its components, (b) organization of replisome transactions and repair, (c) replisome dynamics, and (d) factors influencing the base and sugar fidelity of DNA synthesis.


Introduction
It has been over half a century since the discovery of the first DNA pol ymer ase that Kornber g and collea gues isolated fr om the commensal bacterium Esc heric hia coli in 1956 (Lehman et al. 1958 ).No w ada ys , E. coli is a well-established model organism for studies of DNA synthesis.Its use facilitated many pioneering works that became the cornerstone of the recognized model of DNA replication, including the semiconserv ativ e model of genome duplication described by Meselson and Stahl in their classic paper from 1958 (Meselson and Stahl 1958 ), the discontinuous mechanism of la gging-str and synthesis pr oposed by Okazaki in 1971 (Okazaki et al. 1971 ), but also the v ery natur e of m utations (Luria andDelbrück 1943 , Cairns et al. 1988 ).These findings ar e gener all y univ ersal for all domains of life.Still, more recent disco veries , enabled in particular by the de v elopment of high-r esolution single-molecule imaging, whole genome sequencing (WGS), and cryo-electron microscop y (Cry o-EM), paint a m uc h mor e complex pictur e of E. coli DNA replication than initially thought.This review aims to summarize the available knowledge about E. coli DNA replication with particular interest in the developments of the last decade.

DN A r eplication by the r eplisome
DNA replication is a highly evolutionarily conserved process that r equir es a coordinated action of m ultiple pr oteins r esponsible for the timely and accurate execution of different tasks that can be grouped into three stages: initiation, elongation, and termination (Yao and O'Donnell 2016 ).Bacterial genomes, which are fr equentl y small, circular c hr omosomes (suc h as the ∼4.6 Mb genome of E. coli ), have their replication initiated from a single defined origin site ( oriC ) containing DnaA-binding boxes recognized by the DNA replication initiator protein DnaA (Kaguni 2011, Trojanowski et al. 2018 ).Unlike eukaryotic cells, where origins are licensed for DNA replication well before the S-phase of the cell c ycle (when DN A is r eplicated), in man y bacteria, r eplication may be initiated se v er al times befor e cell division.As a consequence, daughter cells may inherit genomes that already undergo another cycle of replication (the so-called "multifork replication") (Fossum et al. 2007 ).
Cooper ativ e binding of the DnaA molecules at oriC promotes the unwinding of an A:T-ric h DNA fr a gment and loading of the DnaB 6 -DnaC 6 complexes onto each exposed single-stranded DNA fr a gment.The binding of the DnaG primase to the DnaB helicase with DnaC dissociation allows for r eplisome assembl y, completing the last major step of DNA replication initiation (reviewed in Katayama 2017 ).DnaG primase synthesizes ∼10-nt long RNA primers r emov ed at a later step (Zec hner et al. 1992a ).Note that in the simplified model of E. coli DNA replication initiation presented abo ve , only the primary proteins involved wer e described.A mor e compr ehensiv e vie w on the bacterial replication initiation factors can be found in Grimwade and Leonard ( 2021 ).
The elongation stage of DNA replication is carried out by multiple proteins constituting functional complexes called replisomes.The c har acteristics of the E. coli r eplisome, whic h is the focus of this r e vie w, will be described in the following sections.
The last step of DNA replication is termination, which happens when the two r eplisomes a ppr oac hing fr om opposite dir ections conv er ge.E. coli r eplication termination system is centered around the so-called termination region containing 10 terA -J sites that interact with Tus proteins .T hese sites are oriented in such a way that the replication machinery bypasses the first five, but is tr a pped at one of the r emaining fiv e, whic h in turn ar e easily b ypassed b y the r eplisome a ppr oac hing fr om the opposite dir ection.Consequentl y, both r eplisomes become tr a pped in the termination region to avoid chromosome over replication and give the cell time to pr ocess r eplication intermediates (Rudolph et al. 2009(Rudolph et al. , 2013 ) ). Termination is observed predominantly at the four innermost sites , terA -terD (Ivano va et al. 2015, Dimude et al. 2018 ).Inter estingl y, r eplisome tr a pping might be a source of genome instability as recent work utilizing next-generation-based methods [X-seq and END-seq that map the chromosomal positions of Holliday junctions (HJs) and double-stranded breaks (DSBs), respectiv el y] identified ter sites as the region of fr equentl y occurring HJs and DSBs (Mei et al. 2021 ).According to the proposed model, these may arise as a consequence of replication fork stalling and the arrival of another codirectional replisome before fork con vergence , resulting in the displacement of the leading strand, DSB end r esection, and involv ement of homologous recombination (HR) machinery that generates HJs.Ho w ever, these one-ended DSBs cannot be r epair ed until the sister replisome arrives, leading to the accum ulation of r epair intermediates (Mei et al. 2021 ).DNA r eplication termination was r e vie wed r ecentl y in Goodall et al. ( 2023 ).

Esc heric hia coli replisome organization
The DNA elongation step is carried out by the replisomes (reviewed in Yao andO'Donnell 2016 , Xu andDixon 2018 ).The two most essential tasks for the replisome are (1) separation and (2) semiconserv ativ e duplication of the two par ental DNA str ands.Mor e r ecent e vidence suggests that r eplisomes also play a r ole as sensors of obstacles that hinder replication progression and facilitate their repair and tolerance via different interactions (Hawkins et al. 2019, Wolak et al. 2020, Thrall et al. 2022 ).The two major models that explain the spatiotemporal organization of replication describe the replisomes as either mobile complexes that run along DNA like a train on a track or stationary factories anc hor ed at a certain location in the cell with DNA pushed through.In bacterial r esearc h, the dominant vie w is that the replisomes are fixed in space, supporting the factory model (Lemon and Grossman 1998, Brendler et al. 2000, Mangiameli et al. 2018 ).Ho w e v er, both modes were directly observed using live cell imaging in slowgrowing E. coli (Bates and Kleckner 2005, Reyes-Lamothe et al. 2008, Mangiameli et al. 2017, Japaridze et al. 2020 ), leading to the hypothesis that at the beginning of DN A replication, the tw o replisomes ar e coher ed but at some point before termination the two sister replisomes may break apart and travel separately, at least under slo w-gro wth conditions.Recent evidence suggests that the early cohesion of sister replisomes facilitates the establishment of the replication fork and its successful pr ogr ession, while loss of this interaction increases replication fork stalling and the involv ement of r eplication r estart pr otein RecB (Chen et al. 2023 ).The cohesion of the replisomes might, for example, help with the coordination of both replication forks for timely cohesion at the termination site by slowing down one re plicati ve machinery when the other deals with obstacles such as transcription complexes, whic h ar e particularl y abundant at the earl y r eplicating r egion, known to be heavil y tr anscribed in E. coli [see the section "Discussion" in Chen et al. ( 2023 )].
In E. coli , the single re plicati ve polymerase, or the replicase, DNA pol ymer ase III holoenzyme (Pol III HE), is responsible for the lion's shar e of DNA r eplication.Pol III HE is a complex of 10 distinct proteins that can be organized into three subassemblies: the polymer ase cor e (Pol III), the sliding clamp, and the clamp loader complex (CLC; Fig. 1 A) (McHenry 2011 , Yao andO'Donnell 2016 ).An important integral part of the replisome is the helicase DnaB 6 .There is also a plethora of proteins that associate with the replisome either tr ansientl y or for an extended period of time, such as the primase DnaG, accessory DNA pol ymer ases, the singlestr anded-DNA-binding (SSB) pr oteins, topoisomer ases, or some r epair pr oteins suc h as RNase HI.These will be discussed in the following parts of this r e vie w.

The DnaB helicase
The major E. coli re plicati ve helicase is a homohexamer of DnaB subunits encoded by the dnaB gene (r e vie wed in Lewis et al. 2016 , Xu andDixon 2018 ).DnaB 6 translocates 5 → 3 on the laggingstrand template (Fig. 1 A).DnaB is loaded onto single-stranded DN A (ssDN A) as a part of the DnaB 6 -DnaC 6 complex, with the help of DnaA at oriC at the very beginning of DNA replication [see Blaine et al. ( 2023 ) for a r e vie w].Recent crystal structur es of the DnaB 6 -DnaC 6 complex suggest that DnaC binding via its NTD to the CTD of DnaB causes a distortion in the helicase ring.This distortion accumulates when more DnaC units are bound and eventuall y r esults in the helicase opening (Chodav ar a pu et al. 2016 , Chase et al. 2018, Arias-Palomo et al. 2019, Nagata et al. 2020 ).ATP binding in the DnaC ATPase domain stabilizes DnaB open conformation and is important for helicase activation (Arias-Palomo et al. 2019, Puri et al. 2021 ).During loading, DnaB assumes a dilated conformation with a wide central channel, but it can also dynamicall y switc h between dilated and constricted conformations as it tr av els on the ssDNA (Strycharska et al. 2013 ).
The primary role of the helicase is to unwind the DNA duplex and produce single-stranded fragments, which is po w ered b y ATP hydr ol ysis .T he ATP-binding sites positioned a picall y at the replication fork are located in the C-terminal domains (CTDs), whereas the N-terminal domains (NTDs) that form a trimer of dimers participate in pr otein-pr otein inter actions with v arious partners .T he two are connected via linker domains .T he initial insight into the mechanism of translocation was provided thanks to studies involving viral and eukaryotic models (Enemark and Joshua-Tor 2006 , also r e vie wed in Li and O'Donnell 2018 ), and the so-called hand-on-hand model that resembles rope climbing has been proposed for DnaB based on the crystal structure (Itsathitphaisarn et al. 2012 ).According to this model, during translocation, the helicase assumes a spiral staircase-like conformation around ssDNA and binds 12 nucleotides (2 per subunit).ATP hydr ol ysis driv es the movement of the CTD of the 5 -most DnaB subunit to w ar d the 3most subunit, resulting in the unwinding of 2 nucleotides on the la gging str and.For e v ery two mov ements of the CTDs, the dimeric NTDs would mo ve .T he hexameric state of the helicase would be maintained by the presence of flexible linkers.Neither DNA nor the helicase itself rotates during translocation (Itsathitphaisarn et al. 2012 ).The helicase alone in vitro unwinds at the rate of around 30-35 nt s -1 (Kim et al. 1996 ) but is gr eatl y stim ulated by the replisome subunits to support the in vivo rates of replisome pr ogr ession (Chandler et al. 1975 , Mok andMarians 1987 ).Interestingly, single-molecule FRET studies suggest that the external surface of DnaB interacts with the excluded str and, downr egulating its pr ogr ession r ate (Carney et al. 2017 ).
Another role of the helicase is a structural one.In vitro singlemolecule fluor escence micr oscopy studies r e v ealed that DnaB is the most stable component of the replisome, as upon binding it stays associated for up to 30 minutes (Beattie et al. 2017, Spinks et al. 2021 ).In principle, such a long dwell time allows it to remain bound at the replication fork for the whole period of DNA replication.Based on these observations, it has been proposed that DnaB might serve as the anchor that not only organizes replisome assembly but also enables its dynamic and stochastic nature, which has become evident in recent years (Beattie et al. 2017 ).DnaB interacts with DnaA and DnaC, as well as the DnaG primase and the τ subunit of the r eplicase, anc horing both to the replisome.Recent work utilizing genetic and live cell imaging approaches suggests fr equent inter actions between DnaB and the Rep helicase at the replication fork in vivo , allowing for enrichment of the repair helicase near the sites of DNA synthesis, likely to aid barrier r emov al in front of the fork, which is supported by in vitro data (Fig. 1 A) (Brüning et al. 2018, Syeda et al. 2019 ).Another important interacting partner is PriC, a protein involved in replisome reassembly and replication restart (Wessel et al. 2013(Wessel et al. , 2016 ) ).
What happens to the helicase near the termination sites is not well understood.In principle, the helicase could displace the 3 end of the ne wl y synthesized leading str and fr om the opposite fork and continue unwinding.Ho w e v er, bioc hemical studies suggest that in this scenario, DnaB rather encircles double-stranded DN A (dsDN A), which w ould pr eclude an y unwinding (Ka plan and O'Donnell 2002 ).Encircling both DNA str ands r equir es DnaB to be in a nonconstricted state when it is known to unwind more slowl y (Stryc harska et al. 2013 ).Additionall y, DnaC w as sho wn to facilitate helicase unloading in an ATP-dependent manner (Puri et al. 2021 ).It is, ther efor e, possible that slowing down the helicase someho w allo ws it to bind DnaC, which aids its detac hment fr om DNA.Another possibility is that perhaps the two helicase hexamers cannot pass each other and collide head-on, essentially blocking further tr anslocation.Whic h model persists and what exactl y could be the signal for this remains to be understood.

The DnaG primase
The monomeric primase is the product of the dnaG gene (Ro w en and Kornberg 1978 ).DnaG synthesizes 10-12-nt long RNA primers that start DNA replication (Fig. 1 A) (Kitani et al. 1985 ): at least one that primes the leading strand and roughly 2000 that prime each Okazaki fr a gment on the la gging str and.RNA synthesis in E. coli starts at template dCTG sequences (Swart and Griep 1995 ).
DnaG contains three important regions: the zinc-binding domain (ZBD) located at the N terminus, the RNA pol ymer ase domain (RPD), and the CTD (DnaGC) responsible for interaction with the helicase.DnaGC interacts with DnaB NTD, albeit weakly, and this interaction is important to support in vivo rates of RNA synthesis (Johnson et al. 2000, Mitk ov a et al. 2003, Manosas et al. 2009 ), as DnaG on its own binds DNA weakly and is not very efficient (Khopde et al. 2002, Corn et al. 2005 ).This interaction starts during DNA replication initiation when synthesis of the first primer promotes subsequent association of the replicase.Based upon bioc hemical studies, the pr esumed stoic hiometry was 1 DnaB hexamer to 2-3 DnaG monomers (Mitk ov a et al. 2003 ), but structural and biochemical studies suggest that during processive DNA replication, it might actually be 1:1 (Itsathitphaisarn et al. 2012 ), especiall y giv en that m ultiple DnaG monomers bound to the helicase seem to have an inhibitory effect on the replisome (Tanner et al. 2008 ).In the same structur al anal ysis, Itsathitphaisarn et al. ( 2012 ) put forw ar d the hypothesis that the contact between DnaB and DnaG might not necessarily be maintained for the whole period of primer synthesis.As DnaG can be bound to the lagging DNA strand via the interaction with SSB (Yuzhakov et al. 1999 ), it is thus possible that the transient DnaB-DnaG interaction is more important for the deposition of the primase on the template strand and/or quick termination of priming reaction on the one hand, and for the dynamic control of the behavior of DnaB on the other hand, which will be discussed later.It has been suggested that two DnaG copies acting in trans ar e r equir ed for primer synthesis, with one responsible for template recognition via the ZBD and the other carrying out primer synthesis using RPD (Corn et al. 2005 ).
After primer synthesis, DnaG remains bound to the primer and needs to be displaced by the χ subunit of the CLC of the replicase to allow for loading of the processivity factor (Fig. 1 A) (Yuzhakov et al. 1999, Manosas et al. 2009 ).This finalizes the cycle of DnaG in the replisome.

The CLC
The CLC is composed of se v en subunits ( δδ τ (2/3) γ (1/0) ψχ) and plays m ultiple r oles in the r eplisome .T hese functions ar e br ought together via interactions with the five domains (I-V) of the τ subunit.The τ subunit Domain V interacts with the Pol III corether e ar e 2-3 cor es (described later) r esponsible for DNA synthesis and pr oofr eading (Fig. 1 A).One cor e r eplicates the leading str and, while the other one or two replicate the ∼1000-nt long Okazaki fr a gments that together constitute the lagging strand (Okazaki et al. 1971, Zechner et al. 1992b, Tougu and Marians 1996 ).
It was initially assumed that there are two cores that simultaneousl y r eplicate both leading and la gging DNA str ands.Under this scenario, there are two τ subunits, while the third is replaced by its shorter variant ( γ subunit) truncated at the C end, and thus lacking the core-interacting and the helicase-interacting Domains IV and V, r espectiv el y.While both τ and γ subunits ar e pr oducts of the dnaX gene, the shorter γ subunit is a consequence of ribosomal frameshifting taking place during tr anslation (Blink o w a and Walker 1990 ). Ho w e v er, an in vitro study sho w ed that a tri-core replicase could be functional in the replisome (McInerney et al. 2007 ).Soon thereafter, in a series of experiments utilizing fluor escentl y ta gged r eplisome subunits, it w as sho wn that the in vivo stoichiometry of the CLC observed in liv e-cell ima ging is τ 3 δδ ( αεθ) 3 ψχ (Reyes-Lamothe et al. 2010 ).It has been proposed that the purpose of the third τ -bound core might be to facilitate sim ultaneous r eplication of two Okazaki fr a gments (McInerney et al. 2007, Montón Silva et al. 2015 ), but another explanation is that only two cores occupy the sliding clamps, while the third might wait for another clamp to be loaded (Reyes-Lamothe et al. 2010 ).Additionally, single-molecule studies sho w ed that the tri-cor e r eplicase exhibits higher processivity than the two-core isoform (Georgescu et al. 2012 ).This might be related to the fact that Domain IV was also shown to interact with DNA (Jergic et al. 2007 ).Ne v ertheless, a body of data supports the idea that the replisome might contain only two cores, at least under certain conditions (also discussed in McHenry 2011 ).While the γ subunit is not essential for surviv al (Blink ov a et al. 1993 ), it copurifies within the replisome (McHenry 1982, Glover and McHenry 2001, Dohrmann et al. 2016 ) and is also present in other bacteria (Larsen et al. 2000 , Tashjian andChien 2023 ).Additionally, cells lacking the γ subunit show increased UV-sensitivity and defective DNA synthesis by one of the translesion synthesis (TLS) polymerases, DNA pol ymer ase IV (Pol IV), suggesting impaired DNA repair and/or damage tolerance (Dohrmann et al. 2016 ).Consistent r esults wer e also obtained in another bacterium, Caulobacter crescentus (Tashjian and Chien 2023 ).One possibility is that the third core outcompetes Pol IV under stress conditions, causing impair ed dama ge r esponse, but also during the stationary phase, leading to selective disadv anta ge as low-fidelity pol ymer ases ar e str ongl y expr essed during that time and play a role in adaptation (Yeiser et al. 2002, Corzett et al. 2013 ).On the other hand, a threecor e r eplicase might be handy under nutrient-rich conditions, incr easing the pr ocessivity of DNA synthesis during fast gr owth.This suggests that perhaps replicase composition is regulated depending on the cellular state.
The τ subunit Domain IV interacts with the DnaB helicase (Fig. 1 A).The exact interface is not known but is presumed to be dynamic.This interaction is important for processive DNA replication and possibly for sensing the uncoupling of DNA unwinding and synthesis, as the helicase translocation rate depends on τ binding (Kim et al. 1996, Graham et al. 2017 ).Ne wl y obtained biochemical data suggest that a single copy of the τ subunit participates in the DnaB binding (Monachino et al. 2020 ).The strength of this inter action incr eases when DnaG is also bound to the helicase (Monachino et al. 2020 ).A textbook view of this interaction was that it remains stable for extended periods of time during replication extension; ho w e v er, single-molecule studies suggest that this is not the case (discussed later) (Lewis et al. 2017 ).
The τ subunit Domains I-III participate in the k e y CLC acti vity, which is loading the β 2 clamp onto DNA (Fig. 1 A).In particular, Domains I and II form an AAA + interface and exhibit ATPase activity, whereas Domain III is the collar domain that comes into contact with the ψ subunit (Simonetta et al. 2009 ).Domains I-III are common for τ , δ, and δ , although δ and δ do not provide the ATPase activity and do not interact with ψ.Together, they form a pentameric structure in which Domain I create a C-shaped passage for DNA and contact with the clamp, while Domain III form a ring-like collar that sits atop the other domains (Simonetta et al. 2009 ).
The subunits are ordered as follows: δ, γ / τ 1 , γ / τ 2 , γ / τ 3 , and δ (Kazmirski et al. 2004 ).Recentl y, a series of Cryo-EM structur es of the CLC in various conformational states and quaternary complexes with the β 2 clamp and/or DNA have been published, offering insight into the clamp loading cycle (Xu et al. 2023 ).The first step is binding three ATP molecules at the interfaces connecting γ / τ subunits, as well as between γ / τ and δ .ATP binding induces conformational changes, leading to the r eor ganization of the AAA + domains (Hingorani andO'Donnell 1998 , Ason et al. 2003 ).This makes the CLC competent in binding the β 2 clamp, which is the next step of the cycle.Biochemical analysis of the β 2 mutants with a destabilized dimer interface as well as fluor escence pr oximity sensing assay suggest that the ATP-bound CLC activ el y opens closed clamps rather than simply capturing and stabilizing open clamps from the cytosol (Paschall et al. 2011, Douma et al. 2017 ).β 2 binds first via the δ subunit.The gap between δ and δ Domains I then expands through a crab claw-like movement, leading to the opening of β 2 (Xu et al. 2023 ).
As the next step, a primer-template DNA duplex passes through the open channel formed between δ and δ subunits of the pentameric ring (Tondnevis et al. 2016 ).Interestingly, the crystal structure of the CLC in complex with DNA suggests that virtually only the template strand is in contact with the CLC (Simonetta et al. 2009 ).The downstream part of the template strand exits thr ough the ga p between Domains I, II, and III of the δ subunit, wher e the highl y conserv ed loop at positions 276-283 on the exterior surface of the δ collar domain establishes an interaction with the template strand (Chen et al. 2008 ).The exterior surface is positiv el y c har ged and seems to be inter acting not onl y with the template DNA but also with the downstream part of the gapped or nicked nascent strand, providing a structural basis for how the CLC loads the β 2 clamps on such duplexes (Xu et al. 2023 ).Although the structures do not show the position of the highly flexible C-terminal part of the τ subunit, it is known that Domain IV w eakly binds ssDN A and dsDN A (J ergic et al. 2007 ), and thus , it cannot be excluded that this interaction might further stabilize DNA around the CLC.
DNA binding prompts a conformational change with the tightening of the AAA + interface, bringing the arginine fingers close to the ATP molecules and facilitating their concerted hydr ol ysis (Xu et al. 2023 ).This triggers the release of the clamp-encircled DNA from the CLC.
The τ / γ subunit is also in contact with the ψχ tail of the CLC.The two homologs χ and ψ are the products of holC and holD genes, r espectiv el y.They form a dimer with a highly conserved interaction interface, and neither contacts DNA dir ectl y (Gulbis et al. 2004 ).A SAXS structure of the seven-subunit CLC suggests that the ψχ dimer is located close to γ / τ 3 (Tondnevis et al. 2015 ).The Nterminal part of the ψ subunit penetrates the collar domain, interacting with the three τ / γ subunits (Simonetta et al. 2009 ).This not onl y incr eases the str ength of inter actions within the pentamer complex (Olson et al. 1995 ), but also helps the CLC to assume the conformation favored during DNA binding, increasing the affinity by 20-fold (Simonetta et al. 2009 ).The χ subunit interacts with the SSB proteins coating the exposed single-stranded regions of the template DNA strand (Marceau et al. 2011 ) and participates in their remodeling, as suggested based on in vitro FRET assays (Newcomb et al. 2022 ).The interaction of the CLC with SSB stabilizes the complex on primer-template DNA (Newcomb et al. 2022, Xu et al. 2023 ) and is also important for pr ocessiv e Okazaki fr a gment synthesis (Fig. 1 A) (Glover and McHenry 1998 ).The multiple roles of SSB in the replication fork will be discussed later.

The β 2 clamp
The β 2 clamp is a ring-shaped homodimer encircling the primertemplate duplex.The clamp is a homodimer consisting of two 41-kDa proteins encoded by the dnaN gene (Burgers et al. 1981 ).The primary purpose of the β 2 clamp in the replication fork is to increase the speed and the processivity of DNA replication.This is best illustrated by the in vitro biochemical activity of the Pol III cor e, whic h alone replicates ∼20 nt s -1 and 10-20 nt per binding e v ent (Fay et al. 1981 , Maki andKornberg 1985 ).These numbers jump to ∼350-500 nt s -1 and up to ∼2000 nts per binding e v ent when bound to the β 2 clamp (Tanner et al. 2008 ), and to ∼700-1000 nt s -1 and ∼150 000 nts per binding e v ent in the context of the replisome (Mok and Marians 1987, Yao et al. 2009, Tanner et al. 2011 ).All E. coli DNA pol ymer ases wer e shown to increase their processivities 25-400 times upon β 2 binding.
The clamp has a clear pseudo 6-fold symmetry, with the outer circle composed of β-sheets and the ∼30-35 Å inner circle composed of α-helices .T he helices contain many positively charged residues that form electrostatic interactions with DNA that facilitate sliding (Georgescu et al. 2008 ).Both subunits have canonical protein-binding sites in the form of hydrophobic pockets composed partially of DnaN C-terminal residues .T hese pockets bind a variety of DNA-interacting proteins possessing specific clampbinding motifs (CBMs).As pr e viousl y mentioned, during clamp loading, one pr otein-binding poc ket inter acts with the δ subunit of the CLC.During pr ocessiv e r eplication, both ar e normall y occupied by the re plicati ve polymerase and the exonuclease, the α  Sutton et al. 2001, López De Saro et al. 2006, Maul et al. 2007, Pluciennik et al. 2009, Sikand et al. 2021 ).
The network of interactions of the β 2 clamp with its partners is more complicated than singular DNA-binding and proteinbinding sites.For example, the protein-binding pocket also interacts with the single-stranded portion of the primed template, with a possible role during clamp loading (Georgescu et al. 2008 ).Moreover, some clamp-binding proteins, such as DNA polymerase IV, have additional points of contact outside of the hydrophobic cleft (Bunting et al. 2003, Maul et al. 2007, Heltzel et al. 2009, Wagner et al. 2009, Kath et al. 2014 ).A possibility has been raised that these alternative binding sites might facilitate the r a pid exc hange of DNA pol ymer ases during r eplication.Inter estingl y, β 2 m utants carrying mutations within the DNA-binding region have been isolated and shown to somehow affect interactions with Pol III or Pol II and Pol IV (Heltzel et al. 2009, Homiski et al. 2021, Berger and Cisneros 2023 ) and even impair the ability of E. coli to tolerate DNA dama ge (Nanfar a et al. 2016 ).
Apart from increasing the processivity of DNA synthesis, the sliding clamp can also modulate other activities of DNA polymerases, as binding of the β 2 clamp inhibits Pol I stranddisplacement (SD) activity and promotes 5 → 3 exonucleolysis in vitro , possibly to avoid excessive DNA resynthesis during Okazaki fr a gment matur ation (Bhar dw aj et al. 2018 ).As the β 2 clamp also interacts with the ligase, sliding clamps left behind the replication fork might be used by repair enzymes (López de Saro and O'Donnell 2001 , Moolman et al. 2014 ).
Given that around 2000 Okazaki fragments are synthesized in eac h r eplication cycle, the demand for the β 2 clamps far exceeds their cellular le v els (Bur gers et al. 1981 ).As the closed clamp conformation is rather stable (Binder et al. 2014 ), the leftover clamps need to be activ el y unloaded from the dsDNA.Current evidence points in the direction of the δ subunit of the CLC being the unloader, as addition of δ to an in vitro reaction decreases the sliding clamp half-life on DNA from ∼2 hours to around 2 minutes (Yao et al. 1996, Leu et al. 2000 ).These early results are supported by a more recent single-molecule fluorescence microscopy study where it has been shown that shortly after initiation the number of the DNA-bound β 2 clamps increases to eventually reach a constant le v el ( ∼46, whic h is ∼50% of the cellular le v el), maintained until termination, and the half-life of the DNA-bound β 2 clamp was over 3 minutes (Moolman et al. 2014 ).

T he r eplicative cor e
Unlike eukary otes, ar c haea, and man y other bacteria, E. coli Pol III's polymerizing and exonucleolytic proofreading activities are provided b y tw o separate subunits of the re plicati ve core ( α and ε, r espectiv el y, encoded by dnaE and dnaQ genes).The third subunit in the core, θ (encoded by the holE gene), plays a stabilizing role (Fig. 2 ) (Taft-Benz and Sc haa per 2004 ).The last decade has significantly expanded our kno wledge regar ding the structure and interactions within Pol III HE.As pr e viousl y mentioned, during DNA synthesis, α and ε subunits are both bound to the hydrophobic pockets of the β 2 clamp.Much insight into the structural arrangement of the α-ε-β 2 trio came from Cryo-EM studies (Fernandez-Leiro et al. 2015 ).
T he P ol III α subunit (Pol III α) has se v er al domains (Fig. 3 A).At the N terminus, there is the pol ymer ase and histidinol phosphatase (PHP) domain, which in some bacteria (that lack the ε subunit) provides the exon uclease acti vity, although, in E. coli , it has been inactivated during evolution.For this reason, it was belie v ed that PHP mostly plays a structural role, although bioinformatic analysis based on sequence alignments suggested that PHP might be a putative pyrophosphatase (Lamers et al. 2006 , Barros  3 ).The three aspartic acids essential for catalysis, as well as the steric gate residue (His760) located in the vicinity of the 2 carbon of the sugar moiety (star sign), are shown.Major intermolecular contact sites are also marked in (A).The PDB structure 5FKV was used.In (C), the nascent DNA duplex and the incoming nucleotide (dTTP) were modeled based on the PDB structure 3E0D of Taq Pol III. et al. 2013 ).This has been confirmed in subsequent studies, where it was shown that pyrophosphate hydrolysis regulates DNA synthesis rate in vitro and is important for viability and genome stability (Lapenta et al. 2016 ).In the central part of Pol III α are located the palm, the thumb, and the fingers domains (Fig. 3 A).Palm and fingers together participate in creating the active site .T he active site fold of Pol III α, which is a C-family DNA polymerase, is unlike that of eukaryotic B-famil y r eplicases but mor e akin to that of Xfamil y pol ymer ases suc h as human Pols β and λ (P ar asur am et al. 2018 ).Se v er al amino acids within the active site are essential for nucleotide selection and catalysis, including the catalytic aspartic acids at positions 401, 403, and 555 in the palm domain and histidine at position 760 in the fingers domain, but also others, including some more distant, residues responsible for correct positioning of amino acids, electrostatic interactions, and proper closing of the active site (Fig. 3 A-C) (Parasuram et al. 2018 ).The nascent DNA duplex is gripped between the thumb domain (which in the primary structure is nested within the palm domain) and the fingers domain (Lamers et al. 2006, Fernandez-Leiro et al. 2015 ).The fingers domain is longer than in other pol ymer ases but binds DNA loosely, allowing for an unprecedented speed of DNA elongation of ∼700-1000 nt s -1 when bound to the β 2 processivity factor (for comparison, the rate of eukaryotic replication fork pr ogr ession is ∼25-30 nt s -1 ) (Fig. 3 B) (Conti et al. 2007, Sekedat et al. 2010 ).At the C terminus of the fingers domain, there is the β-binding site, and next to it, there is the oligonucleotide binding (OB) domain, and then the τ -binding region and the very C terminus of Pol III α (Fernandez-Leiro et al. 2015 ).
T he P ol III ε subunit (P ol III ε) can be divided into the big Nterminal catalytic domain ( εNTD) and the small C-terminal segment ( εCTS).The catalytic domain contains three conserved Exo motifs (I, II, and IIIe).These motifs contain essential residues D12, E14, D103, and D167 that form a DEDD motif, common for many nucleases (DeRose et al. 2002 ).A fr a gment between Exo I and Exo II interacts with the θ subunit, while Exo III interacts with the thumb domain of Pol III α.At the end of the εNTD, there is the CBM, and the εCTS contains a PHP-inter acting fr a gment connected to the εNTD via a flexible glutamate-rich linker (Fernandez-Leiro et al. 2015 ).Ther efor e, Pol III ε makes two points of contact with Pol III α, which is important for DNA transactions during replication as it allows for partial dissociation of Pol III ε (discussed later, Fig. 2 ).
The plethora of interactions makes the core a tightly bound complex and increases its affinity to the β 2 clamp, especially when also bound to DNA.The Cryo-EM structure revealed that upon DNA binding, the core undergoes a significant conformational change with a major shift of the oligonucleotide (OB) domain that forms another point of contact with the sliding clamp between the pol ymer ase and the exonuclease (Fernandez-Leiro et al. 2015 ).Accordingl y, the cor e-β 2 -DNA complex is m uc h mor e stable than the core with the clamp alone (Naktinis et al. 1996 ).But the core subunits also stimulate each other as the core is in vitro more proficient in both synthesis and exonucleolysis than α and ε alone (described in more detail in Lewis et al. 2016 ).

The ssDNA-binding protein
The SSB protein coats single-stranded fragments of DNA produced by the helicase, thereby protecting them from degradation and pr e v enting formation of DNA secondary structures that could interfere with replication (Fig. 1 A).SSB is a homomeric complex composed of four subunits encoded by the ssb gene .T he Nterminal OB domain of the SSB subunit participates in DNA binding, whereas the C-terminal part contains a long, disordered interdomain linker (IDL) that is important for cooperativity, and the conserved 9-amino-acid-long tail (SSB-Ct) that enables interactions with many different partners (in binding many of which IDL also plays a role; reviewed in Oakley 2019, Bianco 2021 ).
Depending on salt concentration and r elativ e SSB and DNA concentr ations, thr ee SSB binding modes to DNA have been observed that differ in the number of nucleotides per tetramer: SSB 35 , SSB 56 , and SSB 65 (Lohman et al. 1986, Bujalowski and Lohman 1989, Ferrari et al. 1994 ).SSB 35 displays high nearestneighbor binding cooperativity mediated by the interaction between the IDL and the OB domain of the neighboring SSB 35 , and the SSB-DNA complexes form large clusters observed in electron microscopy (Griffith et al. 1984(Griffith et al. , K ozlo v et al. 2015 ) ).On av er a ge, two OB sites ar e involv ed in complex formation.A r ecent study r e v eals the presence of a conserved surface close to the DNA-binding site that interacts with a DNA fragment that "bridges" two tetramers.This interface is important for linking SSB tetramers and thus forming higher-order complexes in SSB 35 binding mode (Dubiel et al. 2019 ).In contrast, the crystal structure of SSB65 shows DNA wr a pped ar ound four SSB subunits and thus occupying all available OB sites in a manner described as a "basketball seam" (Raghunathan et al. 2000 ).These complexes seem to form octamers visualized as beads on DNA in electr on micr oscopy and ar e c har acterized by low binding cooperativity.
Recent investigations utilizing single-molecule force and fluor escence spectr oscopy and optical tweezer methods r e v ealed the mechanisms of DNA wrapping and unwrapping.Binding seems to occur through intermediate states where initially eight and then 17 nt are wrapped, likely mediated by an initial DNA interaction with the W54-R56 cluster (Suksombat et al. 2015, Naufer et al. 2021 ).Wr a pping is linear and goes through SSB 8 , SSB 17 , SSB 35 , and SSB 56 to finall y r eac h the SSB 65 sta ge, while unwr a pping occurs in the opposite direction (Suksombat et al. 2015 ).Notably, singlemolecule studies show that SSB is dynamic on ssDNA and able to not only switch binding modes but also change its position (translocate) (Roy et al. 2007, 2009, Zhou et al. 2011 ).Translocation, which is a fast process, occurs via a mechanism called reptation, a snak e-lik e movement that is typical of long polymers: SSB r emains mostl y bound to DNA, while short 2-5-nt fr a gments ar e unwr a pped and quic kl y r eplaced by another fr a gment of ss-DNA.In the process, a small bulge of unwrapped DNA travels around the SSB tetramer, effectively leading to SSB transloca-tion with respect to DNA (see the Supplementary video in Zhou et al. 2011 ).Other observations suggest that SSBs are initially deposited on ssDNA (e.g.produced by the helicase) and swiftly wr a pped ar ound 65-mers subject to ssDNA availability, but when SSB starts to build up, the binding mode changes to SSB 35, which is a m uc h slo w er pr ocess and pr obabl y mediated by high binding cooperativity (Naufer et al. 2021 ).Based on these observations, a model of SSB dynamics during DNA replication has been pr oposed.When fr ee ssDNA shortens , e .g. due to ongoing DNA synthesis or RecA filamentation, SSBs are first pushed together (due to r a pid tr anslocation), ov ercr owded, and finall y ejected fr om DNA, after which they might quickly reassociate with newly produced ssDNA (Naufer et al. 2021 ).The ov ersatur ation might stimulate the rates of unwr a pping and dissociation, whic h ar e normall y quite low.SSB tr anslocation might be assisted by the movement of the r eplication mac hinery, although an alternativ e explanation is that the replicase activ el y dislocates the SSB (Cerrón et al. 2019 ).
SSB's interactome is extensive and comprises primase; DNA Pols II, III, IV, and V; nucleases such as ExoI, Exo IX, and RNase HI; helicases such as DinG , RecG , or RecQ; primosome proteins PriABC; topoisomerase III; and other DNA repair proteins such as AlkB, RadD, RecO , RecJ , and Ung (Ar ad et al. 2008 ; r e vie wed in Bianco 2021 ).Most of these proteins have an OB-fold that mediates SSB interactions .T hus , SSB can be viewed as an important hub that orc hestr ates r eplisome tr ansactions and facilitates DNA maintenance (Shereda et al. 2008 ).This perspective has been expanded using more recent discoveries in another section of this manuscript.

T he auxiliar y DNA polymerases
DNA pol ymer ase I (Pol I) encoded by the polA gene is not an integral part of the replisome but activ el y participates in DNA replication.The C-terminal segment of Pol I (Klenow fr a gment) contains the pol ymer ase and the 3 → 5 exonuclease, while the N-terminal segment connected via a flexible linker provides the 5 → 3 exonuclease and the endonuclease activities (see , e .g. Lewis et al. 2016 for more details).Pol I synthesizes patches of DNA to replace RNA primers in a process called Okazaki fragment maturation (Fig. 1 A).This allows for the subsequent ligation of Okazaki fr a gments by the DNA ligase .P ol I is also a v ersatile r epair pol ymer ase participating in short-patch repair pathways such as nucleotide excision repair (NER), base excision repair, very short patch repair, and ribonucleotide excision repair (RER) (McDonald et al. 2012 , Robertson andMatson 2012 ).Indeed, Pol I is well-equipped to accomplish these tasks as it is proficient in 3 → 5 exonucleolytic, 5 → 3 exon ucleolytic, endon ucleolytic, and SD acti vities and can perform nic k-tr anslation, ga p-filling, and SD synthesis.On the other hand, the processivity of Pol I is low (15-20 nt synthesized per binding e v ent; Bambar a et al. 1978 ) unless bound to the β 2 clamp (López de Saro and O'Donnell 2001 ), which in turn inhibits its SD activity and pr omotes nic k-tr anslation and earl y ligation in vitro (Bhardwaj et al. 2018 ).This might explain why Pol I is not belie v ed to be the major contributor to the MutSLH-dependent MMR pathway.
An insight into the mechanism of Pol I-mediated Okazaki matur ation was pr ovided in r ecent studies that utilize singlemolecule microscopy, in vitro biochemistry, and structural biology a ppr oac hes (Cr a ggs et al. 2019, P auszek et al. 2021, Botto et al. 2023 ).The first step is the handover of the primer terminus from Pol III to Pol I. How Pol I is recruited to the substrate and whether Pol I binds to a nick or a gap is not clear.In vitro experiments suggest that it might be a nick based on pr ocessiv e Pol III-mediated replication (Botto et al. 2023 ), but the alternative model implies that Pol III might abandon the Okazaki fr a gment befor e it is replicated up to the next primer, thus leaving a gap for Pol I to fill; in vivo experiments are consistent with this idea (Graham et al. 2017 ).Another question is whether Pol I utilizes the β 2 clamp during Okazaki fr a gment matur ation.This seems likel y as the clamp w as sho wn to pr e v ent excessiv e Pol I SD acti vity in vitro (Bhard waj et al. 2018 ), and live-cell imaging revealed that dozens of clamps are bound to DNA in acti vely di viding cells, each for over 3 minutes (Moolman et al. 2014 ).The stead y-state n umber of clamps is r eac hed in less than 10 minutes and r emains so for ov er 60 minutes .T hese clamps are likely left behind by the lagging-strand (and less fr equentl y the leading-str and) pol ymer ase subassembl y at a perfect place to be utilized by Pol I (and also other repair proteins).Ho w e v er, the β 2 clamp does not seem to stimulate Okazaki fr a gment matur ation in vitro (Botto et al. 2023 ).
A structure of Pol I in complex with the template, upstream, and downstr eam str ands sho ws the template is bent b y ∼120 • at the Pol I fingers domain, which leads to partial (1-2 nt) fraying of the RNA substrate, with the unpaired bases interacting with Arg781 and Phe771.This substrate is displaced by the fingers during the Pol I tr anslocation (Cr a ggs et al. 2019, Botto et al. 2023 ).As Pol I replicates up to the end of the primer, it simultaneously cleav es the fla p.It has been shown using FRET microscopy that the fla p can spontaneousl y tr ansfer between the pol ymer ase and the endonuclease (Pauszek et al. 2021 ), which likely facilitates primer nucleolysis.Ho w ever, an in vitro assay that utilized an RNA-primed DN A substrate sho w ed that Pol I is a v ery pr oficient junction nuclease , clea ving on the 3 side of the last ribonucleotide with high specificity (Botto et al. 2023 ).How this specificity is ac hie v ed is not clear, but it stands to reason that Pol I recognizes some kind of additional signal that triggers endonucleol ysis.Inter estingl y, as the endonuclease reaction in (Botto et al. 2023 ) was carried out in the absence of dNTPs, meaning that the pol ymer ase could not translocate, it seems likely that during Okazaki fragment maturation, the cut is introduced before Pol I reaches the end of the flap.T hus , a plausible scenario is that Pol I nicks the RN A-DN A junction at the beginning or during extension of the upstream DNA str and, whic h is accompanied by flap clea vage , and then terminates at the nic k, whic h is subsequentl y ligated by the LigA ligase (Botto et al. 2023 ).
Pols II, IV, and V (encoded by polB , dinB , and umuDC , respectiv el y) ar e the thr ee E. coli DNA pol ymer ases that ar e involv ed in TLS (Fig. 4 A) (see Maslowska et al. 2019 , Fujii andFuchs 2020 for r e vie w).Pol II is a B-famil y pol ymer ase that can bypass abasic sites and acetylaminofluorene adducts (AAF-dG).Y-family Pol IV and Pol V are specialized in dealing with minor groove and major gr oov e lesions, r espectiv el y.For example, Pol IV can bypass alkyl adducts , whereas P ol V performs TLS on UV lesions (Tessman and K ennedy 1994, Na politano et al. 2000, Fujii and Fuchs 2007, Robinson et al. 2015, Wang et al. 2021 ).Unlike P ol V, P ols II and IV are normall y pr esent in the cell at detectable concentrations .T hese levels ar e fiv e times higher for Pol IV ( ∼50 versus ∼250 molecules/cell), but Pol II has a higher affinity for the β 2 clamp (Bonner et al. 1992, Wagner et al. 2000, Sutton 2010 ).Given that Pol II is a high-fidelity, exonuclease-pr oficient DNA pol ymer ase , while P ols IV and V are not, it is possible that one of the cellular roles of Pol II is to limit Pol IV's mutator potential.Additionally, genetic evidence indicates that P ol II ma y serve as a backup replicase when Pol III has trouble continuing the r eplication (Banac h-Orlowska et al. 2005, Fijalkowska et al. 2012 ).
Upon DNA damage, the cellular levels of Pols II, IV, and V can be further ele v ated due to the activ ation of the so-called "SOS" r esponse, whic h r esults in the upr egulation of specific genes (Fer-nández de Henestrosa et al. 2000, Courcelle et al. 2001 ).Other consequences of SOS induction are, for example, activation of NER and HR (r e vie wed in Bell and Ko w alczyko wski 2016 ).The slo wdown of fork pr ogr ession upon DNA damage allows for the binding of RecA protein to DNA.RecA has several cellular functions: it is a pr otein involv ed in HR, but it is also a mediator of the SOS response and SOS-dependent m uta genesis.RecA m ust compete for ssDNA with SSB, which normally coats exposed DNA regions.Because of SSB's high affinity to ssDNA, binding of RecA to DNA is a timel y pr ocess unless aided by other complexes suc h as RecFOR (Morimatsu and Ko w alczyko wski 2003 ).RecFOR can target DN A r epair to ssDNA ga ps, and r ecent studies suggest that this activity might be mediated by RecF interaction with the β 2 clamp, particularly with ones associated at abandoned replication intermediates when the replicase stalls , dissociates , and continues replication downstr eam dama ge (Henry et al. 2023 ).In line with these findings, RecF fr equentl y colocalizes with the replisome (Henrikus et al. 2019 ).Other important factors in SSB displacement are the diffusion of SSB, which leads to exposure of uncoated fr a gments (Roy et al. 2009 ), and SSB-RecA inter action, whic h modulates filament formation (Wu et al. 2017 ).Creation of the nucleoprotein filament (often denoted as RecA * ) promotes proteolysis of LexA, which is a r epr essor of the SOS system that governs the transcription of specific genes such as the umuDC operon encoding Pol V (Shibata et al. 1981, Shinagawa et al. 1988, Patel et al. 2010, Cory et al. 2024 ).lexA and recA genes ar e LexA-r egulated and RecA-der egulated themselv es suc h that SOS induction is suppr essed quic kl y when r eplication fork pr ogr ession is r estor ed, and RecA * filaments are no longer forming.
Pol V is a heterotrimeric protein composed of the UmuC catalytic subunit and two noncatalytic UmuD subunits .P ol V exhibits a strong mutator potential and is subject to an astonishingly complex system of control comprising transcriptional, temporal, spatial, and biochemical elements of the regulation (reviewed in Goodman et al. 2016, Jaszczur et al. 2016 ).Firstly, Pol V is normally undetectable in the cell.After LexA-and RecA-dependent transcription starts, which happens ∼15 minutes after SOS activation, Um uC and Um uD 2 pr oteins ar e quic kl y degr aded by the Lon pr otease , dela ying pr otein accum ulation to ∼45 minutes after SOS activation when the rate of translation overcomes the rate of proteolysis (Gonzalez et al. 1998 ).Secondly, as revealed by the more r ecent single-cell micr oscopy studies, when Um uC and Um uD 2 accum ulate, Um uC is sequester ed at the cell membr ane awaiting Um uD 2 autopr oteol ytic activ ation (the activated dimer is denoted UmuD 2 ), allowing for the assembly of Pol V (UmuD 2 C) ∼1 hour after SOS induction (Robinson et al. 2015 ).Ho w e v er, activ ation of Pol V additionall y r equir es the binding of RecA and ATP, and this active complex is called the Pol V mutasome (Pol V mut).Thirdly, Pol V mut is the slo w est DN A pol ymer ase ( ∼0.29 nt s -1 ), and its pr ocessivity str ongl y depends on SSB ( ∼25 nt per binding e v ent when bound to the β 2 clamp; ∼200 nt per binding e v ent when SSB is also present) (Tang et al. 2000, Pham et al. 2001, Karata et al. 2012 ).Fourthly, as the SOS signal subsides, UmuD binds to UmuD , forming a heterodimer, and then UmuD is degraded by ClpXP, while Um uD and Um uC ar e degr aded by Lon, as already mentioned (Frank et al. 1996, Gonzalez et al. 2000 ).These elements ensure that Pol V expression and action, and therefore its mutator effect, are k e pt to a minim um.Inter estingl y, in the recA730 genetic bac kgr ound, wher e RecA_E38K exists in a constitutiv el y induced state due to the more efficient competition with SSB, most of the regulation is circumvented such that Pol V is constantly expr essed without an y DNA dama ge, pr omoting high le v els of spontaneous m uta genesis, especiall y when combined with a LexA defi-

Other activities near the replisome
Replisomes fr equentl y encounter differ ent insults that block or slo w do wn their pr ogr ession and hav e the potential to affect replication fidelity and genome stability.DNA lesions can be dealt with on the fly using TLS pol ymer ases or by activating the SOS system, which is mediated by the RecA protein.T hese ha ve been described in the pr e vious section.Ho w e v er, an equall y important source of replication obstacles is the never-ending DNA metabolism and maintenance .Replisomes ma y clash with proteins associated in front of the fork or undissociated transcripts, forming structures known as R-loops .T he primary enzymes responsible for the re-mov al of pr otein or RNA adducts fr om DNA ar e Rep helicase and RNase HI, r espectiv el y.Importantl y, both might be viewed as transient, auxiliary components of the replication machinery, as interactions with replisome subunits enrich them at the sites of ongoing replication.
The monomeric Rep helicase translocates 3 → 5 on the leadingstrand template, in contrast to the re plicati ve helicase DnaB (Kor ole v et al. 1997 ).Rep is proficient at displacing DNA-bound proteins such as the RNA polymerase (RNAP) but does not unwind dsDNA (Brüning et al. 2018, Hawkins et al. 2019 ).Another E. coli helicase with partially redundant activity is UvrD, a component of MMR and the NER machinery, as loss of both is lethal under fast gro wth conditions.Ho w ever, only Rep w as sho wn to physically interact with the replisome (Atkinson et al. 2011 ), and unlike in the case of other auxiliary helicases, loss of Rep significantly affects cell growth, for which this interaction is crucial (Atkinson et al. 2011 ).In a recent live cell imaging study, it has been proposed that Rep monomers might occupy all six DnaB subunits, and the interaction is stochastic and dynamic (Syeda et al. 2019 ).Other studies suggest a lo w er occupanc y (Whinn et al. 2023 ).This implies that DnaB may serve as a launching pad for Rep probes, where Rep constantl y surv eils DNA for pr otein r oadbloc ks and performs quick displacement (Fig. 1 A) (Syeda et al. 2019, Whinn et al. 2023 ).We note in passing that UvrD also colocalizes with the replisome to aid protein displacement, but no specific recruitment factors in this context have been identified (Wollman et al. 2024 ).
RNase HI, which will be discussed in detail in the section dedicated to ribonucleotide repair in DNA, is an endoribonuclease that cleav es RNA tr anscripts in R-loops.It has been shown that RNase HI interacts with the C-terminus of SSB, which is important for the stimulation of its activity (Petzold et al. 2015 ).Ho w e v er, mor e r ecent studies r e v ealed that this interaction is responsible for RNase HI colocalization with the replisome (Wolak et al. 2020 ).A mutant strain in which this interaction is eliminated is characterized by slo w ed gro wth and activation of the DN A dama ge r esponse when combined with a Rep deficiency, and this phenotype is dependent on the le v el of ongoing tr anscription, indicating that RNase HI enrichment near the replication fork is important for R-loop r emov al in front of replication fork (Fig. 1 A) (Wolak et al. 2020 ).Importantl y, ther e ar e other enzymes capable of R-loop repair, including DinG helicase which was shown to unwind R-loops (Voloshin and Camerini-Otero 2007 ).DinG is also stimulated by the interaction with SSB (Cheng et al. 2012 ), but whether this protein is deposited at the replisome similar to RNase HI is currently unknown.
Re plisome acti vity leads to the accumulation of topological stress that is relieved by type II topoisomerases acting both in front of and behind the replication fork (Bush et al. 2015 ).Positive supercoiling due to DNA unwinding by the helicase in front of the fork is relaxed by gyrase.Topoisomerase IV (topo IV) may also play a role in this process, but it is essential behind the fork for disentangling daughter c hr omosomes that become catenated due to migration of positive supercoils from the front (Sissi andPalumbo 2010 , Ashley et al. 2017 ).
Topo IV, a tetramer composed of P arC 2 (r esponsible for DNA binding and catalysis) and ParE 2 (ATPase), can work on a variety of substrates, including positive and negative supercoils as well as catenates (Bush et al. 2015 ).Topo IV has been shown using liv e ima ging to colocalize with the structur al maintenance of the c hr omosome (SMC) complex MukBEF that is indispensable for proper positioning and segregation of sister chromosomes (Nicolas et al. 2014, Zawadzki et al. 2015 ).Topo IV-MukBEF interaction is pr obabl y important for decatenation near oriC s in pr epar ation for their subsequent separation.Ho w ever, Topo IV also interacts with the SeqA protein that trails behind the fork where it binds hemimethylated GATC sequences (Fig. 1 A) (Kang et al. 2003 ).SeqA plays multiple roles: it limits overinitiation of DNA replication (Pedersen et al. 2017 ), pr e v ents pr ematur e methylation of DN A b y the Dam methylase, enabling the activity of MMR (Fig. 1 C) (Kang et al. 1999 ), pr omotes earl y cohesion of sister c hr omosomes, whic h is important for proper segregation (Joshi et al. 2013 ), and orchestrates Topo IV action along the replicated DNA (Helgesen et al. 2021 ).The exact mechanism is not clear, but it is possible that Topo IV binds SeqA clusters on the replisome-distal side, where it catal yzes c hr omosome disentanglement.
Gyrase is composed of a single GyrA (DNA binding) and two GyrB (ATP ase) subunits.Gyr ase is not efficient at decatenation, and is thus belie v ed to be primarily responsible for the intro-duction of negative supercoils in front of the fork, at which it is more efficient than Topo IV (Fig. 1 B) (Bush et al. 2015, Ashley et al. 2017 ).A single-molecule study of gyrase distribution in the cell suggests that besides the many gyrase molecules bound across the c hr omosome likel y to maintain steady-state le v els of supercoiling, there is also an enrichment near the replication fork with increased dwell time, suggesting processive action in front of the fork (Stracy et al. 2019 ).Intriguingly, the combined rate of relaxation by gyrase and Topo IV, as observed in these studies, is not sufficient to k ee p up with the rate of DNA replication (Stracy et al. 2019 ), and additional regulatory elements called Replication Risk Sequences have been identified recently.During replication, these GC-rich sequences promote formation of single-stranded gaps on the lagging strand to control supercoil formation (Pham et al. 2024 ).Mor eov er, no specific factors that would recruit gyrase to the r eplisomes hav e been identified, nor is it known whether suc h factors exist; it is possible that the already bound gyrase units are engaged for processive relaxation of topological stress in front of the fork.

The dynamics of the replisome
Multiple lines of evidence suggest that the E. coli replication fork is a dynamic entity that under goes man y tr ansactions involving most of its components .T his includes the dynamic nature of the SSB protein on DNA, the cycles of primer synthesis and clamp loading, and also the exchange of different DNA polymerases at the replication fork and of the replicase holoenzymes themselves.These will be discussed in the following par a gr a phs.

T he helicase-primase-r eplicase axis
DnaB helicase is the central protein of the replisome that links the activities of the DnaG primase and the Pol III holoenzyme.Ho w e v er, the timing of DNA unwinding, priming, and synthesis need to be tightly coordinated to avoid uncoupling and replication failure.An important layer of regulation of DnaB 6 relies on its c ycling betw een the tw o conformational states, dilated and constricted, that vary in properties.For instance, in the dilated conformation, the rate of unwinding is lo w er than in the constricted form (Strycharska et al. 2013 ).Ad ditionally, priming acti vity is stimulated by DnaB 6 in the dilated form when unwinding is also slo w er; constricted DnaB 6 does not support priming (Strycharska et al. 2013, Monachino et al. 2020 ).Likewise, the Pol III τ subunit inter acts str ongl y with the dilated DnaB 6 , significantl y incr easing the rate of unwinding (Monachino et al. 2020 ).
Another layer is primase binding itself.During translocation, DnaG binding sites are constantly disrupted are reformed, suggesting that the primase does not bind the helicase for the whole period or primer synthesis (Manosas et al. 2009, Itsathitphaisarn et al. 2012 ), consistent with the fact that during catalysis, both move in opposite directions.Notably, while analysis of the strength of the DnaB-τ interaction in solution suggests that the free helicase exists predominantly in a state between constricted and dilated, binding of DnaG markedly increases the strength of this interaction, indicating that DnaG binding to the helicase induces a switch to the dilated conformation, which promotes its interaction with the holoenzyme (Monachino et al. 2020 ).As DnaG is likely to be forcibly ejected from the helicase during translocation (Manosas et al. 2009, Itsathitphaisarn et al. 2012 ), and the rate of unwinding is lo w er when there is no CLC (Strycharska et al. 2013 ), it is possible that the pace at which DnaB 6 produces ss-DNA is dynamically adjusted depending on the presence and/or strength of interaction with τ .Indeed, the helicase slows down by 80% in response to leading-str and-r eplicase pausing (Gr aham et al. 2017 ), and conformational transactions are a plausible explanation for this phenomenon.Ho w e v er, the binding of DnaG does not lead to helicase pausing (Monachino et al. 2020 ), and thus, the exact mechanism of how lagging-strand synthesis is coordinated with priming and unwinding remains to be uncovered.

Replicase exchange at the replication fork
Pol III HE is v ery pr ocessiv e, ca pable of synthesizing thousands of kilobases of DNA per binding e v ent at a very high speed in vitro and replicating ∼2.3 Mb (i.e.half of the chromosome per replisome) of DNA in around 40 minutes in vivo .Accordingly, the textbook view of DNA replication has been that the replicase remains steadily bound at the replication fork for the period of DNA replication.Ho w e v er, a body of e vidence gather ed fr om liv e cell ima ging suggests that the DnaB 6 helicase is the only stable element of the r eplisome, r emaining bound at the replication fork for ∼30 minutes (Beattie et al. 2017, Spinks et al. 2021 ).In contrast, P ol III * (i.e .Pol III HE sans the β 2 clamp) at the r eplication fork fr equentl y exchanges with free subassemblies from the cytosol every several Okazaki fr a gments both in vitro and in vivo (Beattie et al. 2017, Lewis et al. 2017 ).As Pol III goes through cycles when it binds the helicase either str ongl y or weakl y (Monac hino et al. 2020 ), and r eplisome pausing e v ery fe w seconds was observed in vitro (Graham et al. 2017 ), it is possible that it is during that conformational switch that Pol III * exchange takes place.
This observ ation r aises se v er al questions.First, is the leading str and r eplication trul y discontinuous?An answer to this pr oblem was provided in a study where replication intermediates from activ el y dividing cells were separated at high resolution using sucr ose gr adients (Cr onan et al. 2019 ).These intermediates wer e a ppr oximatel y ∼80 kb long on the leading strand and ∼1.2 kb long on the lagging strand.T hus , the leading strand is seemingly replicated in a c hemicall y continuous manner, with the possible exception of encounters with different insults that lead to either repriming below the block or replication fork collapse and subsequent r eassembl y, depending on whether the helicase can accommodate them.Ho w e v er, Pol III * is no w kno wn to pause and dissociate from the 3 terminus, which is then picked up by another re plicati ve complex, and therefore the leading-strand replication is also kinetically discontinuous (Graham et al. 2017 ).
Another interesting question is to what extent leading-and la gging-str and r eplication ar e coordinated.A certain le v el of coordination seems necessary as replication forks need to conv er ge timel y, and all ga ps need to be filled as under conditions of fast growth, these nascent DNAs are also templates for the next advancing forks .T his pr oblem pr edominantl y concerns la ggingstr and r eplication, whic h r equir es m ultiple cycles of dissociation, priming, clamp loading, and reassociation.One might expect that the replisome would be regulated in response to these challenges, and yet, no specific signals have been discov er ed, and it seems that the lagging strand has no trouble k ee ping up with the leading str and e v en when priming fr equency is artificiall y alter ed, at least in vitro (Graham et al. 2017 ).This led to the proposal that the leading-and la gging-str and r eplicase subassemblies work independentl y of eac h other.In principle , one ma y hypothesize that the leading-strand pausing and replicase exchange could be the mechanisms that ensure the temporal coordination of both DNA strands .For example , if the la gging-str and cor e had tr ouble completing Okazaki fr a gment synthesis , P ol III * dissociation from the helicase w ould allo w for the replication of the la gging-str and ga p to be completed by this Pol III complex, while another copy of the holoenzyme associates to the helicase and resumes replication (Fig. 5 ).Indeed, according to more recent calculations, the rate at which Pol III * exchanges correlates with the time required for replication of a single Okazaki fr a gment (see the section "Discussion" in Monachino et al. 2020 ).It is also possible that the third core in the holoenzyme participates in la gging-str and r eplication, facilitating a quick switch or even simultaneous replication of two Okazaki fr a gments (Montón Silv a et al. 2015, Beattie et al. 2017, Xu and Dixon 2018 ).Any of these could contribute to diminishing the supposed bottleneck resulting from the lagging-strand replicase cycling.
It is worth noting that in vivo data regarding the ssDNA gap ratio between the leading-and la gging DNA str ands ar e conflicting.In one WGS-based study, in which E. coli cells expressed the CTD of the APOBEC3G deaminase that specifically converts dC to dU in a ssDNA substr ate, a 2-fold str and bias to w ar d the C in the laggingstrand template was observed upon ung deletion (the gene encoding Ung glycosylase that repairs such lesions), suggesting that this strand is more accessible to APOBEC3G (Bhagwat et al. 2016 ).In another investigation, isolated gDN A w as treated with bisulfite, which also deaminates deoxycytidine on ssDNA; here, the sequencing data sho w ed no bias (Pham et al. 2022 ).More studies are needed to determine whether the la gging-str and mac hinery has an y tr ouble k ee ping up with the leading-str and r eplication.

P olymerase switc hing at the r eplication f ork
Apart from the exchange of identical Pol III * complexes, replisomes may also occasionally switc h fr om Pol III-dependent to accessory-pol ymer ase-dependent DNA r eplication (Pols II, IV, and V).K ey e vidence for this phenomenon came from genetic assays showing that that Pol II, IV, and V mutational signatures can be observed in vivo when an exonuclease-deficient mutant of Pol II is expressed, Pol IV is ov er pr oduced, or Pol V is constitutiv el y activ ated (Malisze wska-Tkaczyk et al. 2000 , Kuban et al. 2004, 2005, Banach-Orlowska et al. 2005, Curti et al. 2009 ).Mor eov er, a sim ultaneous defect in pr oofr eading by Pol III and Pol II has a synergistic effect on mutation rates, indicating that Pol II normally repairs err ors intr oduced by the r eplicase (Banac h-Orlowska et al. 2005 ).The effect of the accessory pol ymer ases is exacerbated in strains expr essing m utant Pol III with an incr eased pr opensity to dissociate from the primer terminus (Makiela-Dzbenska et al. 2019 ).These data corr obor ate bioc hemical observ ations (1) that all accessory DNA pol ymer ases inter act with the β 2 clamp (Sutton 2010, Fijalkowska et al. 2012, Yang and Gao 2018, Fujii and Fuchs 2020 ), (2) that Pol II can utilize the CLC for DNA synthesis (Bonner et al. 1992, Kath et al. 2015 ), and (3) that Pols III and IV can be simultaneously bound to the clamp (Indiani et al. 2005 ).
The unique structure of the Pol III core might facilitate the polymer ase exc hange .T he β 2 clamp has two canonical binding sites, one on each subunit, typically occupied by the α and ε subunits (Fig. 2 ) (Jergic et al. 2013 ).Ho w ever, the β-ε interaction is relatively weak, and the exonuclease can fr equentl y dissociate from the clamp while still being bound to the α subunit via its C-terminal part (Toste Rêgo et al. 2013 , Whatley andKreuzer 2015 ).The partial dissociation is possible due to a flexible linker that connects the N and C termini (Fig. 2 ).Increasing the strength of the β-ε interaction, i.e .making P ol III ε less susceptible to dissociation resulted in SOS induction and a defect in TLS, suggesting problems with the efficient repair of lesions and replication fork stalling, because the TLS pol ymer ases hav e tr ouble to effectiv el y substitute for Pol III at the fork (Whatley and Kreuzer 2015 ).Conversely, weakening the inter action incr eases TLS (Chang et al. 2019 ).Ther efor e, the ε subunit serves as a gatek ee per that regulates the access of Pols II, IV, and V to the replication fork (Fig. 4 A and B) (Jonczyk et al. 1988 ,  Kath et al. 2014, 2015, Thrall et al. 2017, Chang et al. 2019, Tuan et al. 2022 ).Inter estingl y, it has been shown that the inter action with SSB might enrich Pol IV near the replication fork, facilitating quick TLS when Pol III stalls (Chang et al. 2022, Thrall et al. 2022 ).On the other hand, single-cell imaging revealed that Pol V foci do not colocalize str ongl y with the replisome upon UV irradiation, suggesting that TLS occurs behind the replication fork (Robinson et al. 2015 ).Similar observ ations wer e made with Pol IV when a different type of DNA lesion was induced (Henrikus et al. 2018 ).Under this scenario, r eplication pr ogr esses after repriming downstream the damage .Hence , repriming with TLS behind the fork and Pol IV-mediated TLS at the fork seem to be two competing mechanisms, with the outcome likely governed by the type of lesion and the strength of the β-ε interaction (Fig. 4 A) (Marians 2018, Chang et al. 2019, Sale 2022 ), and perhaps also simple stochastic competition.Importantly, when the SOS system is constitutively activated, Pol V foci colocalize with the replisome (Robinson et al. 2015 ), but it is unknown whether pol ymer ase switc hing, in this case, is also mediated by Pol III ε dissociation or some other mechanism, especiall y giv en that Pol V activ ation r equir es m utasome assembl y.An alternativ e model of pol ymer ase exc hange that involves a complete dissociation of the Pol III core from the β 2 clamp and the subsequent association of an auxiliary pol ymer ase has been proposed (Zhao et al. 2017 ).

T he r ole of SSB in organizing r eplisome transactions
ChIP-seq anal ysis r e v eals that SSB is ubiquitous on the la gging DNA strand (Pham et al. 2023 ).Such an abundance has several consequences.First, as argued in the pr e vious sections, SSB needs to be fr equentl y r earr anged and/or displaced during r eplication.For example, it is known that SSB-Ct interacts with the χ subunit of the CLC, which is important for its remodeling and for stimulation of its β 2 loading activity (Newcomb et al. 2022 ) that in turn involves a handover of the primed DNA from the DnaG primase, whic h also inter acts with SSB during primer synthesis (Yuzhakov et al. 1999 ).Ho w ever, additional interactions of SSB with P ol III α ha ve been identified (Bianco 2021, McIsaac 2022 ), and it is tempting to speculate that one of their roles might be to facilitate the displacement of SSB during Okazaki fr a gment synthesis (Sokoloski et al. 2016 ).Additionally, the formation of the RecA nu-cleoprotein filament, necessary for SOS activation, also requires SSB displacement, which is usually facilitated by other protein complexes such as RecFOR.Both RecA and RecO interact with SSB (Hobbs et al. 2007, Wu et al. 2017 ).
SSB mediates pr otein-pr otein inter actions via its indispensable C-terminal region as well as the IDL, and besides the ones mentioned abo ve , its interactome encompasses at least another 15 proteins, including SSB itself (Bianco et al. 2017, Bianco 2021 ).How these pr oteins ar e important for DNA r e plication and how the y are affected by the interaction with SSB has been extensively revie wed (Sher eda et al. 2008 ).Her e, instead, we will focus on our understanding of SSB dynamics, which was enabled by novel researc h.In r elation to that, another crucial role of SSB stems from its chemical properties, namely, the propensity to aggregate.It has been shown that SSB tends to form condensates in vitro via a process called liquid-liquid phase separation (LLPS) that is driven by its IDL and SSB-Ct that form multiple weak contacts with neighboring SSB tetramers (Harami et al. 2020 ).These condensates can store a significant amount of proteins, concentrating them at the sites of DNA replication.
A r ecentl y de v eloped super-r esolution ima ging system optimized for use with prokaryotic cells offers a glimpse into SSB dynamics in a living E. coli (Zhao et al. 2019 ).Under unperturbed conditions, SSB forms multiple foci within the cell with particular enrichment at the inner cell membrane, where it binds phospholipids.Ho w e v er, the situation changes upon DNA damage as under these conditions, SSB tends to form distinct spots along the genome, distall y fr om the membr ane (Zhao et al. 2019 ).Formation of the aforementioned liquid condensates in these spots is a likely explanation for this observation (see the section "Discussion" in Harami et al. 2020 ).Accordingly, it stands to reason that SSB would be attracted, for example, to the stalled replication forks by the exposed ssDNA, where these re plisome-pro ximal SSB condensates would deliver different proteins to the sites of DNA damage, facilitating quick damage repair and/or tolerance.Indeed, in recent liv e-cell ima ging studies, under conditions of r eplication str ess, DNA Pol IV, RecG, and PriA w ere sho wn to be enriched near the replication fork, and at least in the case of Pol IV, it is dependent on the interaction with SSB (Chang et al. 2022, Thrall et al. 2022 ).Hence, it can be said that SSB plays a role in the regulation of the DNA dama ge r esponse, not onl y by contr olling access of RecA to DN A, but also b y mobilizing DN A r epair and dama ge toler ance factors that facilitate , e .g. HR, r eplication r estart after fork colla pse, dir ect dama ge r epair, TLS (Sher eda et al. 2008 ), and possibl y bypass of leading-strand-template DNA gaps (Stanage et al. 2017 ).These factors might be deliv er ed not onl y to the sites of ongoing replication but also others, as the damage may occur r andoml y across the genome.
Although LLPS is inhibited by ssDNA, some SSB condensates might also form at the replication fork under physiological conditions (Harami et al. 2020 ).T hus , normally, SSB probably still plays a role in enriching certain factors near the fork, for example, RNase HI (Fig. 1 A) (Petzold et al. 2015, Wolak et al. 2020 ).It is also worth mentioning that SSB sequestration at the cell membr ane is r eminiscent of the mec hanism of Um uC activ ation delay during SOS induction (Robinson et al. 2015 ).T hus , one can speculate that SSB might also play a role in preventing access of certain DNA r epair/dama ge toler ance pr oteins under normal conditions by k ee ping them a wa y fr om the fork.If this wer e true, it might be interesting in the future to understand how the compartmentation of specific proteins is ac hie v ed, as for example, in untreated cells, there is a strong enrichment of RNase HI at the fork (Wolak et al. 2020 ), but m uc h less so in case of P ol IV (T hrall et al. 2017 ).
(T he ca v eat her e is that RNase HI colocalization with the β 2 clamp, but Pol IV with SSB, was assayed in the r espectiv e studies).

Factors influencing the fidelity of DNA replication
Although the three major factors influencing the base fidelity of DNA r eplication hav e been known since the 1990s, the de v elopment of novel methods such as Cryo-EM and live cell imaging, as well as the popularization of deep sequencing techniques, enabled a better insight into their behavior in living cells as well as the structural intricacies involved.Subsequently, some models of how they are triggered, how they act, and what their specificity is had to be r e vised.Ne w players in the ov er all DNA r eplication fidelity were also identified, with the most prominent example being the abundance of ribonucleotides in DNA.

Nucleotide selection
The spatial considerations involved in the selection of nucleotides with the correct base are well-understood; they are common for all DNA pol ymer ases and hav e been well-described (Kunkel andBebenek 2000 , Ludmann andMarx 2016 ).In brief, DNA polymerases select nucleotides according to the rules of Watson-Crick pairing, whic h ar e enfor ced b y the sha pe of the activ e site.Correct pairing ensures that the size of the pair, dictated by the size of the bases and the length of hydrogen bonds, falls within the spatial constraints of the active site.Other important factors are minor gr oov e scanning, i.e. hydr ogen bond formation between the nitrogenous bases and the active site residues, as well as base stacking (Ludmann and Marx 2016 ).Ho w ever, genetic studies reveal that Pol III mutations leading to a mutator phenotype are sometimes located in amino acids that do not make direct contact with DNA or the incoming nucleotide.For example, mutations in Pol III Serine 759 are thought to cause impaired closing of the fingers domain over the palm domain during catalysis, which might contribute to incr eased m uta genesis observ ed in vivo for example due to improper geometry of the active site in the closed conformation (P ar asur am et al. 2018(P ar asur am et al. , Vaisman et al. 2021 ) ). Apart from that, the propensity of DNA polymerases to mispair deoxyribonucleotides also depends on the sequence context and their capability to extend the mismatch.For example, in strains deficient in both pr oofr eading and MMR, tr ansitions ar e m uc h mor e frequent than transversions, and they are more likely to occur at the 5 N A/G C3 + 5 G T/C N3 sites (Lee et al. 2012, Niccum et al. 2018 ).Additionally, template or primer misalignment are common sources of insertions and deletions (Kunkel andBebenek 2000 , Niccum et al. 2018 ).

Intrinsic pr oofr eading
The textbook view of the transition from DNA synthesis to proofr eading upon mismatc h cr eation is that the mispaired nucleotide induces structur al c hanges that lead to the primer terminus being passed from the polymerase to the exonuclease .T hese were thought to be mediated by the movement of Pol III ε due to the presence of the flexible linker.Ho w e v er, m ultiple lines of evidence support the hypothesis that, at least in the case of Pol III, the exon ucleolytic acti vity is regulated thermodynamically.First, using the single-molecule optical tweezers a ppr oac h, it has been shown that the pol ymer ase and the exonuclease activities are independent.The pol ymer ase pr efer entiall y binds the primer-template junction, whereas the exonuclease preferentially binds ssDNA and can similarly cleave mismatched primers as well as free ss-DNA.While the rate of initiation by the pol ymer ase does not depend on force, the rate of initiation by the exonuclease is forcedependent (Naufer et al. 2017 ).These results indicated that it is the instability of the mismatched primer rather than duplex distortion that initiates pr oofr eading because the pol ymer ase pr eferably binds a stable primer.Second, although the distance between the pol ymer ase and the exon uclease acti v e site is gr eater than 7 nm (Ozawa et al. 2013 ), a Cryo-EM structure of the Pol III core in pr oofr eading mode r e v ealed that when Pol III switches to proofr eading, the cor e under goes v ery little structur al c hanges, with a small movement of the thumb domain away from DNA, a shift of the exonuclease to w ar d DN A, and DN A itself anc hor ed to the internal surface of the β 2 (Fernandez-Leiro et al. 2017 ).Using FRET, it has been shown that the time r equir ed to switc h fr om pol ymerization to exonucleolysis does not depend on the strength of the β-ε interaction, suggesting that it is not broken during switch (Park et al. 2018 ).Taken together, these data corr obor ate the model that the primer instability drives proofreading, and molecular dynamics simulations supported by in vitro biochemistry offer a glimpse into how its journey to the exonuclease is guided by the fine motions of the Pol III core (Dodd et al. 2020 ).Interestingly, the mismatches leading to transversion mutations are repaired by proofr eading mor e efficientl y than those r esulting in tr ansitions (for r e vie w, see B ębenek and Ziuzia-Graczyk 2018 ).

P olymerase exc hange and extrinsic pr oofr eading: differ ential fidelity of the leading and lagging DNA strands
DNA pol ymer ase exc hange at the r eplication fork might hav e a profound impact on the fidelity of DNA replication.It is known that in wild-type E. coli , the lagging DNA strand is replicated with a higher fidelity than the leading strand (Fijalkowska et al. 1998, Lee et al. 2012 ).T he differences ha ve been ascribed to the frequent dissociation of the r eplicase fr om the terminal mismatch during DNA synthesis (Fig. 4 B).As the lagging DNA strand is replicated discontinuousl y, the dissociation e v ents ar e assumed to be mor e frequent on this strand.Upon dissociation, reassociation of an exonuclease-pr oficient DNA pol ymer ase via its exonuclease activity (i.e. a pr oofr eader suc h as Pol III ε or Pol II exo) would likely r esult in r emoving the mismatc h (Banac h-Orlowska et al. 2005 ), contributing to the high fidelity of la gging-str and synthesis.Conv ersel y, the binding of a low-fidelity, pr oofr eading-deficient DNA pol ymer ase suc h as P ol IV or P ol V would result in the extension of the mismatch (Fig. 4 B), leading to strand-bias reversion and the la gging str and being mor e m uta genic (Malisze wska-Tkaczyk et al. 2000 , Kuban et al. 2004Kuban et al. , 2005 ) ).The latter phenomenon has been called "spontaneous mutator activity" or "untargeted SOS mutagenesis" to distinguish it from the DNA damage-induced mutator activity, which is not strand-biased as it is dependent on the presence of DNA dama ge, whic h can occur on both template strands (Gawel et al. 2002 ).
The model has been confirmed in further experiments utilizing the Pol III α "antimutator" alleles (such as dnaE915 ) with an incr eased r ate of dissociation from DN A (Maslo wska et al. 2018, Makiela-Dzbenska et al. 2019 ).One might expect that an increased c hance of r eplicase dissociation should hav e little effect on the la gging-str and r eplication fidelity as this strand is normall y r eplicated in a discontinuous manner.Ho w e v er, it could influence the m utation r ates on the leading strand because pol ymer ase exchange now becomes an important replication fidelity factor on this strand as well.T hus , in strains expressing the "antimutator" alleles, due to the more frequent dissociation of Pol III from the mispair, an antimutator effect has been observed compared to the wild-type strain because the proofreading-proficient Pol III ε and Pol II can now more efficiently remove terminal mismatches not only from the lagging but also from the leading DNA strand.Consistent with the increased access of low-fidelity proofreadingdeficient DNA pol ymer ases to DNA r eplication, ov er pr oduction of Pol IV or constitutive activation of Pol V in dnaE915 strains resulted in a mutator phenotype, which was then observed for both leading and la gging DNA str ands (Maslowska et al. 2018, Makiela-Dzbenska et al. 2019 ).These findings wer e r eca pitulated by other labor atories: pr efer ential access of Pol IV to the la gging-str and replication has been observed in vitro (Yuan et al. 2016 ), and wholegenome sequencing a ppr oac hes hav e shown that Pol V pr efer entiall y r eplicates the la gging str and in constitutiv el y SOS-induced strains (Niccum et al. 2018, Faraz et al. 2021 ).

MMR system
The last line of defense against mismatched nucleotides is the MMR system.The crude model of E. coli MMR comprising MutS, MutL, MutH, UvrD, SSB, an exonuclease, a DNA pol ymer ase, and a ligase is well-established, but has been expanded and r e vised owing to more recent single-molecule and Cryo-EM studies.MMR is initiated by MutS 2 , which forms a circular dimer responsible for scanning the DNA for mismatch-induced conformation disruptions (or indel-producing looped-out nucleotides).The presence of a mismatch induces conformational changes that make MutS 2 competent for binding MutL (Fernandez-Leiro et al. 2017 ).ATP binding allows it to act as a clamp loader and recruit the dimeric MutL 2 clamp (Yang et al. 2022 ).Both can move bidirectionally on the DNA (Hasan and Leac h 2015 ).The curr ent model is that the MutS 2 clamp does not stay at the mismatch site but diffuses , and thus , multiple MutS 2 dimers can be engaged by a single mismatch (Hao et al. 2020 ).Ad ditionally, li ve-cell imaging r e v ealed that MutL 2 is more abundant at the mismatch site than MutS 2 , suggesting that multiple MutL 2 dimers are loaded per repair e v ent (Elez et al. 2012 ).This finding is in line with r ecent e vidence showing that MutS 2 -MutL 2 interaction is dynamic and that MutS 2 is not r equir ed for MutH activity, suggesting that its primary role is to load MutL 2 and arguing against the general consensus that MutS 2 and MutL 2 form a stable complex (Yang et al. 2022 ).
MutL 2 does, ho w e v er, r ecruit and form a searching complex with the MutH restriction endonuclease , which clea ves the unmethylated strand at the 5 side of the recognized GATC sites (Liu et al. 2016 ).Near the cleav a ge site, MutL 2 ca ptur es the UvrD helicase that unwinds DNA 3 → 5 , and thus exposes the template for resynthesis .T he general consensus was that the ssDNA fr a gment displaced by the helicase is cleaved by one of the cellular exonucleases, but the recent single-molecule biochemical study suggests that this is not necessarily the case (Liu et al. 2019 ).The exposed ga pped fr a gment of the c hr omosome is cov er ed by SSB, and then DNA Pol III is engaged to resynthesize the DNA patch.The size of the patch is governed by the distance between GATC sites and can be as big as 1 kb.An additional role of MutL 2 is protecting the 3 end of the resected daughter strand from the premature activity of Pol III (Borsellini et al. 2022 ).
Despite these advances, the model of methyl-directed MMR is far from complete, and important questions remain to be answered.First, it is not entirely clear how MMR is recruited to DNA.It is known that both MutL 2 and MutS 2 interact with the β 2 clamp, with MutS containing two clamp-binding sites in its N-and Cterminal domains (López De Saro et al. 2006 ).Disruption of the C-terminal motif, which confers strong interaction, does not affect repair, but mutations in the weaker N-terminal-binding site impair MMR activity.Based upon these observations, it has been initiall y pr oposed that when Pol III dissociates from the clamp, MutS 2 binds and scans for mismatches directly behind the fork (López De Saro et al. 2006 ).Ho w ever, as β 2 clamps are more abundant on the la gging str and, this model would imply that MMR might be more efficient on one strand than on the other, for what there is no supporting genetic evidence (Niccum et al. 2018 ).Additionall y, further bioc hemical studies suggested that mutations in the N-terminal motif result in less stable pr otein, whic h is the likely cause of the hypermutator phenotype (Pluciennik et al. 2009 ).For the same reason, an alternative model suggesting that replisome-bound clamps serve as launching pads for MutS 2 also seems unlikely (Hasan and Leach 2015 ).One explanation is that there are no specific recruiters but given that MMR activity hinges on MutH cleaving a mismatc h-pr oximal GATC site before it is methylated by Dam, and Dam action is normally delayed by SeqA, one might entertain the idea that Dam and/or SeqA could contribute to MMR deposition.Indeed, there are some data from bacterial 2-hybrid system suggesting that Dam and MMR proteins interact in vivo (Tsai 2019 ).Another important problem is how the directionality of DNA unwinding is ac hie v ed, assuming that UvrD can only translocate 3 → 5 , but MutH moves bidirectionally after being loaded by MutS 2 .It stands to reason that there must be a signal that precludes UvrD translocation when it is bound 5 to the mismatch, as in this case, DN A w ould be unpr oductiv el y unw ound aw ay from the mismatch.At last, it is unknown how the substrate is handed over to the DNA polymerase for resynthesis.As the gap might be quite big, it is gener all y thought that the polymerase is assisted by the processivity clamp.T hus , β 2 could be a likely suspect as it interacts with both Pol III and MutS and MutL.Ho w e v er, as ar gued befor e, MutS 2 inter action with β 2 is not important for this activity, and disrupting the MutL 2 -β 2 interaction results in only a mild mutator phenotype, suggesting that β 2 clamp is not important for substrate handover (Pillon et al. 2015 ).
In contrast to proofreading, E. coli MMR mainly repairs transitions rather than transversions.Correct base pairing, proofreading, and MMR ensure the high fidelity of DNA replication at one mutation per ∼10 10 paired bases or per ∼2 × 10 3 replication cycles (Sc haa per 1993 , Lee et al. 2012 ).It is worth mentioning that MMR's capacity to repair replication errors is limited, and when Pol III's pr oofr eading activity is se v er el y impair ed, MMR might easily become overwhelmed (Fijalkowska andSchaaper 1996 , Niccum et al. 2018 ).

DNA damage
DNA damage is an important source of genetic instability.The sour ces of DN A damage can be grouped into endogenous (such as o xidati v e str ess) and exogenous (e.g.UV irr adiation, exposur e to alkylating agents or antibiotics).Genetic studies utilizing mutation accumulation (MA) assays in strains lacking major DNA repair or damage tolerance pathways reveal that when cells are not exposed to exogenous str ess, the onl y major source of mutations is o xidati v e str ess, leading to the formation of 8-oxo-G (Foster et al. 2015, Bhawsinghka et al. 2023 ), with a minor effect of spontaneous cytosine deamination (Bhagwat et al. 2016 ).
Exogeneous damage is frequently mutagenic as it might lead to activation of the TLS polymerases (Robinson et al. 2015, Henrikus et al. 2018 ).TLS is not a r epair mec hanism but r ather a toler ance mechanism, as the lesion is not removed but bypassed at the cost of fidelity.T hus , instead, cells usuall y attempt to suppr ess TLS by engaging other pathways that are normally error-free (e.g.NER or HR) (Naiman et al. 2016 ).In contrast to the spontaneous mutator phenotype, dama ge-induced m uta genesis is not str and-biased, as lesions might occur on both DNA strands (Gawel et al. 2002 ).Re-centl y, a mec hanism of how cells tr ansientl y ele v ate their m utation rates to facilitate the emergence of antibiotic resistance in E. coli has been described (Gutierrez et al. 2013, Pribis et al. 2019, Zhai et al. 2023 ).Exposure to a subinhibitory concentration of cipr ofloxacin, a DSB-inducing a gent, launc hes a cascade of signaling that leads to the consecuti ve acti vation of the RecA-dependent SOS response, the ppGpp-dependent stringent response, and the RpoS-dependent gener al str ess r esponse.Inter estingl y, this elaborate network of signaling is induced only in a subset ( ∼20%) of cells that show ele v ated le v els of r eactiv e oxygen species (ROS) after SOS induction, and this subpopulation of cells exhibits a hypermutator phenotype (400 times over the remaining cells) (Pribis et al. 2019, Zhai et al. 2023 ).T hus , while other cells remain stable, these "gambler" cells undertake the risk of the stress phenotype to help de v elop antibiotic r esistance .T he role of ROS in this process is in line with the previously described mutator effect of inactivation of oxidative damage repair (Foster et al. 2015 ) and has been lately receiving more appreciation (Qi et al. 2023 ).Importantly, as other work shows that ppGpp binding to the RNAP might pr omote its bac ktr ac king (Kamartha pu et al. 2016 ), the pr oposed model suggests that m uta genic r epair might be concentrated at the sites of heavy tr anscription, possibl y driving the evolution of str ongl y expr essed genes (see the section "Discussion" in Zhai et al. 2023 ).

The randomness and the nonrandomness of genomic mutations
T he abo v e-described model of how cells risk a m utator phenotype to adapt to harsh environmental conditions raises the question of whether the m uta genesis observ ed in living cells is trul y r andom.Ther e ar e man y facets to this pr oblem, and m uc h insight was pr ovided from MA assays together with WGS analyses, as those studies look at mutations in living cells at a genome-wide scale.From the analysis of the rates of mutations in coding versus noncoding r egions, synon ymous v ersus nonsynon ymous m utations, codon usa ge, the r ate of terminating mutations, and the rate of deleterious mutations, it has been concluded there is little selective pressur e a part fr om slight bias to w ar d noncoding regions in the DN A of wild-type E. coli (Lee et al. 2012 ).Ho w e v er, the data gather ed from MMR-or exo-strains hinted at the possibility of there being a selectiv e pr essur e to acquir e m utator phenotype suppr essors (Niccum et al. 2018 ).Whether this might be evidence for nonrandomness of mutations depends on the definition of randomness in this context, as one might not necessarily expect that mutations in MMR-or exo-strains would be concentrated at the genomic regions containing genes where such suppressors are more likely to occur.This is hard to investigate, but mutations were 20% more likely to occur in coding genes in MMR-or exo-strains (Niccum et al. 2018 ).
It is worth mentioning that MA assays r e v eal significant differences in mutation rates across different sequence contexts, which might r eflect differ ential pr opensities of particular DNA pol ymerases to create and extend mismatches and/or indels, which were documented in the past in various in vitro studies, but also possibl y pr efer ences of the r epair mec hanisms as well (Lee et al. 2012, Niccum et al. 2018, 2020 ).There is also a growing body of evidence that in certain bacteria, r eplication-tr anscription conflicts might be a significant source of genome instability (Lang and Merrikh 2018 ).The studies performed in E. coli ar gue a gainst this, as there was little correlation between mutation rates and the level of transcription, gene orientation with respect to replication, or the transcribed and the nontranscribed strand (Lee et al. 2012, Foster et al. 2021 ).T hus , it seems that unlike in many pathogenic bacteria, TC repair is not a major source of genome instability in E. coli (Foster et al. 2021 ).
T here is , ho w ever, an interesting observation that in E. coli and some other pr okaryotes, m utation r ates acr oss the c hr omosome form a wave-like pattern symmetrical around the origin of replication (Niccum et al. 2019 ).One important cause of this pattern is fluctuations of the dNTP pools during DNA synthesis, as changes in dNTP concentrations are known to affect the rate of synthesis and, thus, the chance for error correction.Indeed, there is a body of evidence showing that perturbances in dNTP pools have a significant impact on replication fidelity (Ahluwalia and Sc haa per 2013 , Sc haa per and Mathe ws 2013 , Gawel et al. 2014, Maslowska et al. 2015, Tse et al. 2016 ).Mutation rate distribution was also changed upon loss of nucleoid-binding proteins HU and Fis, indicating that the fidelity is compromised when the c hr omosome is highl y structur ed.Another source of genome instability seems to be replication fork pausing or stalling, as Rep deficiency disrupted the pattern (Niccum et al. 2019 ).

Ribonucleotide incorporation into DNA by the replicase
Apart from correctly pairing the nitrogenous bases, during DNA synthesis, DNA pol ymer ases face the equally important task of selecting nucleotides with the right sugar (Joyce 1997 , Brown andSuo 2011 ), complicated by the fact that the cellular concentrations of the ribonucleotides can exceed those of the corresponding deo xyribon ucleotides over a 100-fold (Bennett et al. 2009, Ferr ar o et al. 2009, Nic k McElhinn y et al. 2010, Cerritelli and Cr ouc h 2016 ).It was only in the 2010s that the extent of ribonucleotide incorporation during DN A replication w as fully appreciated.It is now known that ribonucleotides are the most common noncanonical nucleotides in DNA and are three orders of magnitude more frequent than mismatches (Nick McElhinny et al. 2010, Yao et al. 2013, Vaisman and Woodgate 2015 ).In E. coli, an ywher e between 200 and 600 ribonucleotides are incorporated during a single replication cycle (Cronan et al. 2019, Zatopek et al. 2019 ).
In most DNA pol ymer ases, sugar selection relies on a single amino acid residue termed the "steric gate".The steric gate is a bulky amino acid whose side chain localizes in the vicinity of the 2 carbon of the nucleotide's sugar moiety, creating a steric hindr ance whene v er a ribon ucleotide positions itself at the acti ve site (Joyce 1997 , Brown andSuo 2011 ).For example, in E. coli Pol III, the steric gate is His760 (Fig. 3 C) (P ar asur am et al. 2018 ).Other bulky amino acids such as tyrosine , phenylalanine , and glutamic acid are frequently used as steric gates (Joyce 1997 , Brown andSuo 2011 ).Mutating the steric gate of a r eplicase usuall y r esults in a catal yticall y dead v ariant, wher eas the steric gate mutants of other pol ymer ases, suc h as the TLS pol ymer ases, exhibit significantl y incr eased ribonucleotide incor por ation r ates due to the very low sugar selectivity.Some DNA pol ymer ases, but seemingl y not in E. coli , r el y on a "steric fence" formed by the protein backbone for ribose discrimination (Brown et al. 2010, Cavanaugh et al. 2010, 2011 ).Additionally, it has been shown that a part fr om the steric gate, E. coli Pol IV also has a polar filter residue that draws the 2 -OH of the ribonucleotide close to the protein surface, creating a clash (Johnson et al. 2019 ).Ribonucleotide incor por ation r ates v ary significantl y among DNA pol ymer ases, with the r eplicases usually exhibiting higher sugar discrimination.E. coli Pol III incor por ates r oughl y one rNMP per 2300 nucleotides in vitro (Yao et al. 2013, Sc hr oeder et al. 2015 ).DNA Pol IV has a rather high sugar selectivity, comparable to that of Pol III, while Pol V shows poor sugar discrimination (Vaisman et al. 2012 ).
Another significant source of ribonucleotides in DNA, although tr ansientl y, is primer synthesis by primases.Primers constitute r oughl y ∼1% of the lagging strand and, in E. coli , ar e r emov ed via a Pol I-and RNase HI-dependent pathway(s) described earlier.
Notabl y, RNA tr anscripts may occasionall y inv ade DNA behind the RNAP, and if not r emov ed, they can prime the DNA synthesis (Pomerantz and O'Donnell 2008 ).This is especially true in bacteria where replication and transcription are not temporally separ ated, and r eplisomes ar e likel y to encounter transcription mac hinery.As alr eady mentioned, in some bacteria, such as Bacillus subtilis or Salmonella typhimurium , but not in E. coli , replicationtr anscription conflicts ar e r esponsible for the hyperm utator phenotype and contribute to the de v elopment of antibiotic resistance (Lang and Merrikh 2018 ).Ho w e v er, sometimes, after tr anscription, the RNA transcripts do not disengage from DNA, forming so-called R-loops.R-loops are naturally used for the initiation of replication of ColE1-type plasmids (Naito andUchida 1986 , Subia andKogoma 1986 ), but ov er all, their pr esence in genomic DNA has deleterious consequences.In bacteria, they can initiate replication from noncanonical origin sites, leading to constitutive stable DNA replication (cSDR) (Asai andKogoma 1994 , Kogoma 1997 ).cSDR is initiated at the heavil y tr anscribed r egions of DNA suc h as rrn (encoding rRNAs) and significantl y c hanges the r eplication profile in E. coli (Maduike et al. 2014, Dimude et al. 2015 ).cSDR is oriC -independent and strong enough to maintain DNA synthesis in the absence of DnaA.Uncontrolled replication in both directions would lead to frequent fork collapse, potentially creating m ultiple single-str anded r egions pr one to DSBs.Another source of DSBs is the creation of R-tracts when R-loops are incorporated into DNA as primers (K ouzmino v a et al. 2017 ).Additionall y, Rloops may cause replication fork stalling and, if not displaced, requir e r eplication r estart abov e dama ge in bacteria (K ouzmino va and Kuzminov 2021 ).Many proteins are involved in R-loop repair, most notably RNase HI.The repair and significance of R-loops in bacteria and eukaryotes were extensively reviewed in Brickner et al. ( 2022 ) and McLean et al. ( 2022 ).

RNase HI
Hybrid ribonucleases (RNases H) are nonsequence-specific endoribonucleases that recognize and cleave RNA parts in the RN A:DN A hybrids (Cerritelli and Cr ouc h 2009, Tadok or o et al. 2009, Hyjek et al. 2019 ).They belong to the RNase H-like superfamily that also comprises HIV-1 reverse transcriptase, transposases, HJ r esolv ases, and other nucleases (Majorek et al. 2014 ).RNase HI encoded by the rnhA gene is responsible for cleaving RNA strands in the RNA:DNA hybrids (or hybrids of a DNA strand and a chimeric strand that contains DN A and RN A fr a gments).It is a single-subunit protein with a catalytic domain that pr efer entially binds the RN A:DN A hybrids, and this pr efer ence is ac hie v ed thanks to (a) the interactions of four 2 -OH groups of the RNA strand with the protein chain and (b) a forced conformational change of the DNA strand sugar puckers to a B form, unfavorable for RNA (Hyjek et al. 2019 ).Because of this binding mode, it has been proposed that RNase HI r equir es at least four consecuti ve ribon ucleotides in the RNA fr a gment; howe v er, cleav a ge of a c himeric DNA str and containing a patc h of thr ee ribonucleotides has been reported (Haruki et al. 2002, Reijns et al. 2012 ).Therefore, it seems that at least two ribonucleotides are required at the 5 side and at least one at the 3 side of the cleav a ge site for hydr ol ysis to occur (5 -rN-rN-/-rN-3 , Fig. 6 A) (Reijns et al. 2012(Reijns et al. , Łazowski et al. 2023 ) ).Unlike the eukaryotic counterparts, E. coli RNase HI (as in most other bacteria) cleav es RNA distributiv el y.Additionally, it has been proposed that on a substrate mimicking an Okazaki primer (RNA patch with a 3 overhang on the opposite DNA strand), E. coli RNase HI can work as a pr ocessiv e exoribonuclease (Fig. 6 A) (Lee et al. 2022 ).
As already mentioned, the primary function of RNase HI is the r emov al of R-loops.As R-loops were likely used to initiate DNA replication in ancient life , RNase HI pla yed a crucial role in this process, and examples can be seen today.Replication of bacterial plasmids possessing ColE1-type ori is initiated from a transcript processed by RNase HI (Naito andUchida 1986 , Subia andKogoma 1986 ). RNase HI was also reported to be r equir ed for the completion of replication of E. coli DN A b y processing over-replicated genome fr a gments near the termination site (Wendel et al. 2021 ).Esc heric hia coli RNase HI interacts with SSB and colocalizes with the r eplisome, possibl y to r emov e R-loops in fr ont of the r eplicase (Petzold et al. 2015, Wolak et al. 2020 ).RNase HI activity may also provide a secondary pathway for primer r emov al during the Okazaki fr a gment pr ocessing (Ogawa and Okazaki 1984, Balakrishnan and Bambara 2013, Randall et al. 2019, McLean et al. 2022 ).

RNase HII
RNase HII is specialized in identifying single ribonucleotides (Fig. 6 A).The specificity of the enzyme is lar gel y dictated by the presence of an absolutely conserved tyrosine residue in the active site .T his tyrosine , on the one hand, inter acts (along with pr otein bac kbone r esidues) with the 2 -OH of ribose and, on the other hand, positions itself such that the nucleotide located on the 3 side of the ribonucleotide in the chimeric strand cannot contain a 2 -hydroxyl group due to an imminent steric clash (Hyjek et al. 2019 ).Hence, RNase HII is a junction ribonuclease that cleaves at the 5 side of the RN A-DN A junction in the substrate (5 -/-rN-dN-3 , Fig. 6 A).Bacterial RNase HII is a monomer (encoded by the rnhB gene) and gener all y r equir es RN A-DN A junction unless its pr eferr ed metal ion Mg 2 + is swapped with Mn 2 + , in which case RNase HII can cleave distributi vely lik e RNase HI (Rychlik et al. 2010 ).
Bacterial RNase HII does not participate to a great extent in Rloop repair; ho w ever, it has been shown in E. coli that loss of RNase HII exacerbates gr owth r etardation caused by the lack of RNase HI activity, suggesting some, perha ps secondary, r ole in this process (K ouzmino v a et al. 2017 ).In an y case, the primary function of RNase HII is the r emov al of single ribonucleotides incor por ated by DNA pol ymer ases during r eplication (Sc hr oeder et al. 2015 , Vaisman andWoodgate 2015 ).Loss or impairment of ribonucleotide r emov al has no phenotypical manifestation in E. coli , unlike in eukaryotes (Williams and Kunkel 2022 ).

RER
The term "ribonucleotide excision repair" can be used in a broader sense to describe any pathway engaged in removing single or m ultiple ribonucleotides fr om DNA, as e vidence shows that mor e than one exists in both bacteria and eukaryotes (Vaisman andWoodgate 2015 , Williams andKunkel 2022 ).In general, ribonucleotide repair requires four stages: (a) nucleic acid incision 5 from the ribonucleotide, (b) resynthesis of DNA, (c) removal of the r edundant r esynthesized ribonucleotide-containing fr a gment of the nucleic acid, and (d) ligation of DNA.The canonical RER pathway depends on the activity of RNase HII and was first described in yeast (Sparks et al. 2012 ).According to the current model of RER in E. coli , an incision is follo w ed b y SD synthesis by Pol I (Vaisman and Woodgate 2015 ) (Fig. 6 B).T hen, P ol I's innate flap endon uclease acti vity r emov es the r emaining fla p. Alternativ el y, Pol I has been shown to use its 5 → 3 exonuclease to perform nicktranslation synthesis in vitro as an alternative mechanism of RER (Vaisman and Woodgate 2015 ).Ho w e v er, E. coli str ains expr essing Pol I mutants deficient in different activities are proficient in RER, suggesting that Pol III and other cellular exonucleases can replace Pol I during resynthesis and excision ste ps, respecti vely (Vaisman et al. 2014 ).
Unlike in eukaryotic cells, RNase HII does not interact with the β 2 clamp, and thus, it has been initially assumed that RER occurs passiv el y via diffusion and r andom binding of RNase HII to DNA.Unexpectedly, it has been shown that E. coli RNase HII interacts with the RNAP, making RER a transcription-coupled (TC) r epair mec hanism similar to NER (Hao et al. 2023 ).Based on Cryo-EM structures, RNase HII seems to sit in front of the RNAP, activ el y scanning the transcribed template DNA strand for ribonucleotides, while the pol ymer ase acts as a motor in this context (Fig. 6 B).Key to this mechanism is the observation that both sense and antisense strands are actively transcribed in E. coli (Hao et al. 2023, Tjaden 2023 ).Manipulating the le v el of tr anscription and disrupting the RNAP-RNase HII interaction interface gr eatl y diminishes RER, although it does not eliminate it, showing that most ribon ucleotide re pair in vivo occurs via TC-RER (Hao et al. 2023 ).
Perhaps one of the most surprising discoveries in the field was that RER activity might influence the final replication fidelity (in terms of base selection) in E. coli .Strains expressing the steric gate mutant of the low fidelity Pol V (Pol V_Y11A) or its orthologue subcloned from an integr ativ e-conjugativ e element R391 (Pol V R391 _Y13A) exhibit lo w er m utation r ates than the isogenic strains with wild-type polymerases (Vaisman et al. 2012, Walsh et al. 2019 ).Inactivating RNase HII-dependent RER partially restored the Pol V-dependent mutator phenotype (McDonald et al. 2012, Walsh et al. 2019 ).This led to the hypothesis that excessi ve ribon ucleotide incorporation and the subsequent enhanced RER activity can lead to the r emov al of not onl y ribonucleotides but also adjacent mismatched deo xyribon ucleotides during RERpatc h r esynthesis (McDonald et al. 2012, Vaisman et al. 2012, 2013, 2014, Walsh et al. 2019 ).Ther efor e, RER seems to be an important player influencing genetic stability not only by pr e v enting c hr omosome instability but also by contributing to low mutation rates.

NER as the alternative RER pathway
As RNase HII deletion in E. coli led to only a partial r estor ation of the Pol V-dependent m uta genesis in SOS-induced strains expressing Pol V_Y11A, it was theorized that in its absence, backup RER pathways could partially compensate for the lack of RNase HII-RER.This led to the identification of two backup RER pathways in E. coli dependent on the activities of RNase HI andNER proteins (McDonald et al. 2012 , Vaisman et al. 2013 ).The role of RNase HI was anticipated as Pol V_Y11A can in vitro incor por ate pol yribonucleotide str etc hes, a known substr ate for RNase HI.Howe v er, the involv ement of NER was mor e sur prising because the ribonucleotide-induced helix distortion was not expected to be sufficient for UvrAB (DeRose et al. 2012 ).Based upon the structural analyses and molecular dynamics simulations, it was suggested that the change in electrostatic interactions between the additional 2 hydroxyl group of the ribose ring and the surface residues of UvrB might contribute to the rifbonucleotide being recognized as a lesion (Cai et al. 2014 ).Additionally, in vitro studies suggest that lesion recognition might be affected by the ribonucleotide being mismatched or by the presence of more ribonucleotides in the vicinity (Vaisman et al. 2013 ).In contr ast, pr oofr eading of ribonucleotides by the replicase, suggested by in vitro studies in yeast, seems to make a limited contribution to ov er all RER in E. coli (Łazowski et al. 2023 ).This is consistent with other observations suggesting that pr oofr eading by Pol III ε is mostly driven by primer instability, which is probably not the case if the terminal ribonucleotide is corr ectl y pair ed (see section Intrinsic proofreading ).

Strand specificity of RER
Recentl y, activ e site m utants of DNA pol ymer ases c har acterized by increased ribonucleotide incorporation rates were used together with mutational spectra analyses and WGS-based Hydr ol ytic Ends sequencing method to study RER efficiency on both DN A strands (Łazo wski et al. 2023 ).Surprisingly, it has been shown that RNase HII activity during RER is more important during leading-str and RER, wher eas on the la gging str and, it cooper ates with backup RER pathwa ys .T his division of labor between the tw o DN A str ands is conserv ed fr om normal r eplication to SOSinduced m uta genesis (Łazowski et al. 2023 ).One possible explanation for these observations is related to RNase HII involvement in TC-RER.Most heavily transcribed genes are co-oriented with replication, meaning that RNAP is biased toward moving along the leading-strand template (Goehring et al. 2023 ).Indeed, it seems that the ov er all tr anscription le v el of the leading-str and template is slightly higher than that of the lagging-strand template [personal observations based on the analysis of RNA-seq data in Tjaden ( 2023 )].Another possibility stems from the more puzzling discovery that RNase HI stimulates the repair of single ribonucleotides on the lagging strand during normal replication despite the lack of such activity in vitro (Łazowski et al. 2023 ).To explain these findings, it has been proposed that RNase HI might be indir ectl y involv ed in the r epair of primer-pr o ximal single ribon ucleotides by virtue of its participation in Okazaki fr a gment maturation.Although RNase HI is not required for the removal of RNA primers, ther e is e vidence that on a substr ate mimic king primed DNA duplex, it might act as a pr ocessiv e exoribonuclease (Lee et al. 2022 ).As Pol I was shown to resynthesize primers by making an initial nick at the RN A-DN A junction and then replicating up to the nick (Botto et al. 2023 ), it is possible that shortening the primer disables this mechanism, and thus allows Pol I to resynthesize bigger chunks of DNA, accidentally repairing some ribonucleotides in the process (see the section "Discussion" in Łazowski et al. 2023 ).That would make Okazaki fr a gment matur ation-associated RER the first, although not the major, mechanism of ribonucleotide r epair stim ulated by the action of RNase HI, specificall y on the la gging str and.

Concluding remarks
For many years, E. coli has proven to be an excellent model organism for studying bacterial physiology, and the results obtained have often become a starting point for the investigations of analogous processes in other bacterial families or eukaryotes.In particular, the mechanisms determining the fidelity of replication, so universal for all organisms , ha ve been extensively studied using E. coli as a r epr esentativ e example .T his is possible due to the r elativ e simplicity of the E. coli re plicati ve apparatus, emphasized by the presence of the single re plicati ve polymerase.For instance, unlike Gr am-positiv e bacteria or eukaryotic cells, where differences in the fidelity of the leading-and la gging-str and r eplication might be related to the presence of multiple replicases, with E. coli , one is able to dismiss this problem and focus on the determinants of replication fidelity (in terms of both base and sugar selection) that stem from the basic principles of DNA r eplication, suc h as continuous versus interrupted synthesis of the two DNA strands.This simplicity that we describe is also portrayed in the emerging model of the stochastic nature of the E. coli replisome , where , o verall, v ery little contr ol is imposed ov er its elements .T hese elements can fr equentl y and fr eel y exc hange in the cytosol; mor eov er, the n umber of re plicati ve cores or even the active polymerase at the replication fork may change, and some data indicate that replication of the tw o DN A strands might not be coordinated, in principle allowing for engagement of two separ ate r e plicati ve complexes for DN A replication.P erhaps the most striking example is the observed loss of one daughter c hr omosome when its r eplication cannot be finalized due to the presence of a gap in the template, as E. coli cells were shown to k ee p di viding regardless of the gap (Laureti et al. 2015 ).
At the same time, in certain ar eas, ther e is a surprising level of complexity, for example, in the tight regulation of the activity of the most err or-pr one E. coli DNA pol ymer ase , P ol V, and in how differ ent pr oteins (suc h as SSB, RNAP, or the helicase) are exploited as either sensors , motors , or mobilizers of DNA repair and damage tolerance factors.Indeed, in this particular problem, it seems that E. coli leaves very little to chance, putting to rest the long-standing dispute about macromolecular crowding in the cellular space .T he elaborate network of signaling that ultimately leads to the emergence of antibiotic resistance upon DNA damage is a remarkable example of how our understanding of these processes has practical relevance to clinical r esearc h.
Despite many years of work, we are still searching for new data that would enable a thorough understanding of the mechanism of DNA replication and the factors determining replication fidelity or controlling the stability of genetic material, and some identified players r equir e mor e in-depth anal ysis.Among suc h issues, ther e is , for example , the question of how cells ac hie v e timel y conv er gence of the forks if there really is so little coor dination betw een leading-and la gging-str and synthesis, or if, and how, the number of r eplicativ e cor es at the r eplication fork is r egulated.Ther e ar e also some open questions that br oadl y concern the factors ensuring genome stability: the intricacies of MMR recruitment and action remain to be uncovered, as do the mechanisms ensuring timely delivery of the repair and tolerance factors upon DNA damage.If anything, the pr ogr ess of the last decade, not onl y r egarding r esearc h itself but also the availability of novel high-resolution methods, lets one be optimistic.

Figure 1 .
Figure 1.Esc heric hia coli replication fork and its surroundings .T he replisome (A) and the major sites of activity in front of (B) and behind (C) the fork are magnified.(A): The multisubunit replisome consists of the DnaB helicase, the DnaG primase, tetrameric SSB proteins, the CLC, and 2-3 identical Pol III cor es r esponsible for r eplication.These subassemblies ar e inter connected b y a plethor a of inter actions.Ho w e v er, mor e r ecent e vidence shows that re plisome mak es contact with man y other pr oteins, so as to enric h and r ecruit them to the site of DNA synthesis .T he pur pose of these inter actions is to facilitate DNA damage repair or tolerance (e.g. an interaction between the C-terminal tail of SSB with DNA Pol IV is shown in the picture), Okazaki fr a gment matur ation (DNA Pol I), or r emov al of fr equentl y encounter ed r eplication obstacles suc h as pr oteins (inter action between the DnaB helicase and the Rep helicase) and RNA transcripts (interaction between the C-terminal SSB tail and RNase HI).Howe v er, the mec hanism of r ecruitment of some of them (e.g.mismatch repair proteins or Pol I) remains to be uncovered.(B): Movement of the replication fork increases the topological stress related to the accumulation of positive supercoils, which in front of the fork are relaxed by the gyrase.(C): The supercoils may migrate behind the fork by virtue of replisome rotation, leading to the entanglement of sister chromosomes, which is resolved predominantly by topoisomerase IV.Topo IV is tempor all y and spatially separated from the ongoing replication by the SeqA protein filaments that protect the immediate vicinity of the replisome, delaying DNA disentanglement and methylation.

Figure 2 .
Figure 2. Structure of the Pol III re plicati ve core bound to the β 2 clamp.The N-terminal parts of the α polymerizing subunit and the ε proofreading subunit occupy the two canonical protein-binding sites in the dimeric β clamp.The C-terminal part of the ε subunit is located close to the α subunit PHP domain, and the two fr a gments of ε are connected via a flexible glutamine-rich linker.The θ subunit of the core is nested in-between ε and α.The C-terminal fr a gment of the CLC τ subunit is also shown.The PDB structur es 5FKV and 5M1S wer e used.Modeller was used to model the possible position of the missing ε internal linker.

Figure 3 .
Figure 3. Structure of the α polymerizing subunit of E. coli Pol III.Compared to Fig. 2 , the structures have been rotated clockwise by 90 • .The primary (A) and the ternary (B) structures are shown along with the close-up view of the active site (C); the positions of DNA and other core subunits are visible (see Fig.3).The three aspartic acids essential for catalysis, as well as the steric gate residue (His760) located in the vicinity of the 2 carbon of the sugar moiety (star sign), are shown.Major intermolecular contact sites are also marked in (A).The PDB structure 5FKV was used.In (C), the nascent DNA duplex and the incoming nucleotide (dTTP) were modeled based on the PDB structure 3E0D of Taq Pol III.

Figur e 4 .
Figur e 4. T he fate of lesions and mismatches at the replication fork.(A) When the replicase encounters a DNA lesion, specialized TLS DNA pol ymer ases ar e r ecruited to help r eplicate past the dama ge .T his might ha ppen either at the r eplication fork via pol ymer ase switc hing, or behind the replication fork after polymerase dissociation and subsequent repriming downstream the lesion.(B) Mismatches introduced by the replicase might be r emov ed by the intrinsic pr oofr eading activity provided by Pol III ε.Alternativ el y, partial dissociation of Pol III allows for recruitment of auxiliary DNA pol ymer ases, with the outcome (excision or extension of the mismatch) depending on the associated polymerase (high-or low-fidelity).

Figure 5 .
Figure 5.A theoretical model of replisome dynamics during leading-and la gging-str and r eplication.(A): Pol III * is tightly bound to the helicase, allowing for fast and pr ocessiv e sim ultaneous unwinding and r eplication of both DNA str ands.(B): When DnaG primase is r eady, it disembarks fr om the helicase and proceeds to synthesize a primer, guided by the interaction with SSB.The loss of DnaB-DnaG interaction induces conformational changes in the helicase that destabilize the interaction with Pol III * .Pol III core subassembly on the leading strand pauses, and the helicase slows down.(C): A new DnaG subunit binds to DnaB 6, and a new Pol III * is recruited.On the lagging strand, the CLC of the new Pol III * displaces the primase, loads the β 2 clamp, and the synthesis of the next Okazaki fr a gment begins.On the leading strand, the second core binds to the β 2 clamp left behind by the pr e vious Pol III * and r esumes r eplication.If necessary, the pr e vious P ol III * ma y quic kl y finish the r eplication of the pr e vious Okazaki fr a gment.

Figure 6 .
Figure 6.Ribonucleotide excision repair (RER) in E. coli .(A) Substrate specificity of RNases HI and HII.RNase HI recognizes polyribonucleotide (3 + nt) tr acts, or RNA str ands hybridized to DNA.T he clea v a ge site is at least two ribonucleotides from the 5 end of the tract.RNase HI cleaves the RNA patch distributiv el y, pr oducing a wide range of products of differing lengths.Additionally, E. coli RNase HI was shown to work as a pr ocessiv e exoribonuclease in the presence of a 3 overhang in the opposite strand, i.e. on a substrate mimicking an Okazaki primer.In contrast, RNase HII recognizes single ribonucleotides in a DNA strand.This enzyme is a junction endoribonuclease that pr efer entiall y cleav es at the 5 side of the RN A-DN A junction.(B) Model of E. coli transcription-coupled RER.RNase HII rides in front of the RNAP, scanning the transcribed strand for ribonucleotides .Clea vage of the template strand at the 5 side of the ribonucleotide probably leads to transcription termination and RNAP dissociation, upon which DNA polymerase I resynthesizes a fragment of DNA.The flap is removed by Pol I's innate flap endonuclease (FEN) activity .Lastly , DNA ligase I r emov es the remaining nic k. (C) Str and specificity of RER in E. coli .RNase HII-de pendent RER is the primary pathway of ribon ucleotide r emov al with a particularl y important role on the leading strand.In contrast, on the lagging strand, it cooperates with other RER pathways that are dependent on the activities of RNase HI and NER.Additionally, RNase HI stim ulates the r e pair of single ribon ucleotides on the la gging str and, and the possible mec hanism involv es RNase HI participation in Okazaki fr a gment matur ation (mor e details in text).Notabl y, under certain conditions RER may stimulate the repair of mismatched deo xyribon ucleotides, contributing to the high fidelity of DNA replication.